Ligand (act-4-l) to a receptor on the surface of activated cd4+ tcells

ABSTRACT

The invention provides ligands and fragments thereof to a receptor on the surface of activated CD4 +  T-cells. An exemplary ligand is designated ACT-4-L-h-1. Preferred fragments include purified extracellular domains of ligands. The invention also provides humanized and human antibodies to the ligand. The invention further provides methods of using the ligand and the antibodies in treatment of diseases and conditions of the immune system. The invention also provides methods of monitoring activated CD4 +  T-cells using the ligands or fragments thereof.

CROSS-REFERENCE TO RELATED APPLICATION

Copending application U.S. Ser. No. 08/147,784, filed Nov. 3, 1993,describes related subject matter and is incorporated by reference in itsentirety for all purposes.

TECHNICAL FIELD

This invention relates generally to the isolation and characterizationof a ligand (ACT-4-L) to a receptor on the surface of activated CD4⁺T-cells. This invention also provides antibodies to the ligand, andmethods of using the ligand and the antibodies for monitoring and/ormodulating immune responses.

BACKGROUND OF THE INVENTION

Immune responses are largely mediated by a diverse collection ofperipheral blood cells termed leukocytes. The leukocytes includelymphocytes, granulocytes and monocytes. Granulocytes are furthersubdivided into neutrophils, eosinophils and basophils. Lymphocytes arefurther subdivided into T and B lymphocytes. T-lymphocytes originatefrom lymphocytic-committed stem cells of the embryo. Differentiationoccurs in the thymus and proceeds through prothymocyte, corticalthymocyte and medullary thymocyte intermediate stages, to producevarious types of mature T-cells. These subtypes include CD8⁺ T cells(also known as cytotoxic/suppressor T cells), which, when activated,have the capacity to lyse target cells, and CD4⁺ T cells (also known asT helper and T inducer cells), which, when activated, have the capacityto stimulate other immune system cell types.

Immune system responses are elicited in several differing situations.The most frequent response is as a desirable protection againstinfectious microorganisms. However, undesired immune response can occurfollowing transplantation of foreign tissue, or in an autoimmunedisease, in which one of a body's own antigens is the target for theimmune response. Immune responses can also be initiated in vitro bymitogens or antibodies against certain receptors. In each of thesesituations, an immune response is transduced from a stimulating eventvia a complex interaction of leukocytic cell types. However, theparticipating cell types and nature of the interaction between celltypes may vary for different stimulating events. For example, immuneresponses against invading bacteria are often, transduced by formationof complexes between an MHC Class II receptor and a bacterial antigen,which then activate CD4⁺ T-cells. By contrast, immune responses againstviral infections are principally transduced by formation of MHC ClassI/viral antigen complexes and subsequent activation of CD8⁺ cells.

Over recent years, many leukocyte cell surface antigens have beenidentified, some of which have been shown to have a role in signaltransduction. It has been found that signals may be transduced between acell-surface receptor and either a soluble ligand or acell-surface-bound ligand. The amino acid sequences of leukocyte surfacemolecules comprise a number of characteristic recurring sequences ormotifs. These motifs are predicted to be related in evolution, havesimilar folding patterns and mediate similar types of interactions. Anumber of superfamilies, including the immunoglobulin and nerve growthfactor receptor superfamilies, have been described. Members of the nervegrowth factor receptor family include NGFR, found on neural cells; theB-cell antigen CD40; the rat OX-40 antigen, found on activated CD4⁺cells (Mallet et al., EMBO J. 9:1063-1068 (1990) (hereby incorporated byreference for all purposes); two receptors for tumor necrosis factor(TNF), LTNFR-1 and TNFR-II, found on a variety of cell types; 4-1BBfound on T-cells; SFV-T2, an open reading frame in Shope fibroma virus;and possibly fas, CD27 and CD30. See generally Mallet & Barclay,Immunology Today 12:220-222 (1990) (hereby incorporated by reference forall purposes).

The identification of cell-surface receptors has suggested new agentsfor suppressing undesirable immune responses such as transplantrejection, autoimmune disease and inflammation. Agents, particularlyantibodies, that block receptors of immune cells from binding to solublemolecules or cell-bound receptors can impair immune responses. Ideally,an agent should block only undesired immune responses (e.g., transplantrejection) while leaving a residual capacity to effect desirableresponses (e.g., responsive to pathogenic microorganisms). Theimmunosuppressive action of some agents, for example, antibodies againstthe CD3 receptor and the IL-2 receptor have already been tested inclinical trials. Although some trials have shown encouraging results,significant problems remain. First, a patient may develop an immuneresponse toward the blocking agent preventing continuedimmunosuppressive effects unless different agents are available. Second,cells expressing the target antigen may be able to adapt to the presenceof the blocking agent by ceasing to express the antigen, while retainingimmune functions. In this situation, continued treatment with a singleimmunosuppressive agent is ineffective. Third, many targets fortherapeutic agents are located on more than one leukocyte subtype, withthe result that it is generally not possible to selectively block oreliminate the response of only specific cellular subtypes and therebyleave unimpaired a residual immune capacity for combating infectiousmicroorganisms.

Based on the foregoing it is apparent that a need exists for additionaland improved agents capable of suppressing immune responses,particularly agents capable of selective suppression. The presentinvention fulfills these and other needs, in part, by providing a ligand(ACT-4-L) to a receptor localized on activated human CD4⁺ T-lymphocytes.

SUMMARY OF THE INVENTION

The invention provides purified ACT-4-L ligand polypeptides. Thepolypeptides have a segment between 5-160 contiguous amino acids fromthe amino acid of an exemplified ACT-4-L ligand designated ACT-4-L-h-1.The polypeptides usually exhibit at least 80% sequence identity to theACT-4-h-L-1 sequence and often share an antigenic determinant in commonwith the ACT-4-L-h-1 ligand. Usually, the polypeptides comprise anextracellular domain.

The invention also provides purified extracellular domains of ACT-4-Lligands. These domains comprise at least five contiguous amino acidsfrom the full-length ACT-4-L-h-1 extracellular domain. Some of theseextracellular domains are full-length. Other extracellular domains arefragments of full-length domains. Some extracellular domainsspecifically bind to the ACT-4-L-h-1 ligand. Other extracellular domainsspecifically bind to an exemplified receptor of ACT-4-L-h-1, thereceptor being designated ACT-4-h-1. Some extracellular domains consistessentially of a domain possessing a particular functional property, forexample, the capacity to specifically bind to the ACT-4-h-1 receptor.Some extracellular domains inhibit in vitro activation of CD4⁺ T-cellsexpressing the ACT-4-h-1 receptor on their surface. Other extracellulardomains stimulate in vitro activation of such T-cells. Any of the aboveextracellular domains may further comprise a linked second polypeptidesuch as the constant region of an immunoglobulin heavy chain.

The invention also provides an ACT-4 receptor polypeptide consistingessentially of a domain that specifically binds to the ACT-4-L-h-1ligand.

The invention further provides antibodies that specifically bind toACT-4-L-h-1, preferably to an extracellular domain thereof. Preferredantibodies are humanized antibodies and human antibodies. The antibodieshave a variety of binding specificities. For example, some humanizedantibodies specifically bind to the ACT-4-L-h-1 ligand on the surface ofa B-cell so as to inhibit activation of the B cell. Other antibodiesstimulate activation of B-cells. Other antibodies specifically bind tothe ACT-4-L-h-1 ligand on the surface of a B-cell so as to inhibit thecapacity of the B-cell to activate CD4⁺ T-cells.

In another aspect, the invention provides pharmaceutical compositions.The pharmaceutical compositions comprise a pharmaceutically activecarrier and an agent that specifically binds to an extracellular domainof the ACT-4-L-h-1 ligand.

The invention further provides methods of suppressing an immune responsein a patient having an immune disease or condition. The methods compriseadministering an effective amount of a pharmaceutical compositioncomprising a pharmaceutically active carrier and an agent thatspecifically binds to the ACT-4-L-h-1 ligand. Preferred agents aremonoclonal antibodies, ACT-4-L ligand polypeptides and ACT-4 receptorpolypeptides. The invention provides other methods of suppressing animmune response in which the agent competes with the ACT-4-L-h-1 ligandfor specific binding to the ACT-4-h-1 receptor.

The invention also provides methods of screening for immunosuppressiveagents. The methods comprise contacting an ACT-4-L-h-1 ligandpolypeptide with a potential immunosuppressive agent. Specific bindingbetween the ACT-4-L-h-1 ligand polypeptide and the agent is detected.The specific binding is indicative of immunosuppressive activity.

The invention also provides methods of monitoring activated CD4⁺T-cells. The methods comprise contacting a tissue sample from a patientwith an ACT-4-L ligand polypeptide that specifically binds to anextracellular domain of the ACT-4-h-1 receptor. Specific binding betweenthe ACT-4-L ligand polypeptide and the tissue sample is detected toindicate the presence of the activated CD4⁺ T-cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Two-color staining of peripheral blood lymphocytes to analyzeexpression of ACT-4-h-1 on different cell types.

FIG. 2: Kinetics of ACT-4-h-1 expression on alloantigen-activated CD4⁺T-cells. MCF=Mean channel fluorescence.

FIG. 3: Kinetics of ACT-4-h-1 expression on tetanus-toxoid-activatedCD4⁺ T-cells.

FIG. 4: Kinetics of ACT-4-h-1 expression on PHA-activated CD4⁺ T-cells.

FIG. 5: cDNA (upper) and deduced amino acid sequence (lower) ofACT-4-h-1. The Figure indicates the locations of an N-terminal signalsequence, two possible signal cleavage sites (vertical arrows), twoglycosylation sites (gly), a transmembrane domain (TM), a stop codon anda poly-A signal sequence.

FIG. 6: Construction of expression vector for production of stabletransfectants expressing ACT-4-h-1.

FIG. 7: FACS™ analysis showing expression of ACT-4-h-1 on stabletransfectants of COS-7, Jurkat and SP2/O cell lines.

FIG. 8: Fusion of an ACT-4-h-1 extracellular domain with animmunoglobulin heavy chain constant region to form a recombinantglobulin.

FIG. 9: Schematic topographical representation of recombinant globulinformed from fusion of an ACT-4-h-1 extracellular domain with animmunoglobulin heavy chain constant region to form a recombinantglobulin.

FIG. 10: cDNA sequence and predicted amino acid sequence of ACT-4-L-h-1.Boxed regions designate a transmembrane domain, four glycosylation sitesand a poly-A signal.

DEFINITIONS

Abbreviations for the twenty naturally occurring amino acids followconventional usage (Immunology—A Synthesis, (E. S. Golub & D. R. Gren,eds., Sinauer Associates, Sunderland, Mass., 2nd ed., 1991) (herebyincorporated by reference for all purposes). Stereoisomers (e.g.,D-amino acids) of the twenty conventional amino acids, unnatural aminoacids such as α,α-disubstituted amino acids, N-alkyl amino acids, lacticacid, and other unconventional amino acids may also be suitablecomponents for polypeptides of the present invention. Examples ofunconventional amino acids include: 4-hydroxyproline,γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine,O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine,5-hydroxylysine, ω-N-methylarginine, and other similar amino acids andimino acids (e.g., 4-hydroxyproline). In the polypeptide notation usedherein, the left-hand direction is the amino terminal direction and theright-hand direction is the carboxy-terminal direction, in accordancewith standard usage and convention. Similarly, unless specifiedotherwise, the lefthand end of single-stranded polynucleotide sequencesis the 5′ end; the lefthand direction of double-stranded polynucleotidesequences is referred to as the 5′ direction. The direction of 5′ to 3′addition of nascent RNA transcripts is referred to as the transcriptiondirection; sequence regions on the DNA strand having the same sequenceas the RNA and which are 5′ to the 5′ end of the RNA transcript arereferred to as “upstream sequences”; sequence regions on the DNA strandhaving the same sequence as the RNA and which are 3′ to the 3′ end ofthe RNA transcript are referred to as “downstream sequences”.

The phrase “polynucleotide sequence” refers to a single ordouble-stranded polymer of deoxyribonucleotide or ribonucleotide basesread from the 5′ to the 3′ end. It includes self-replicating plasmids,infectious polymers of DNA or RNA and non-functional DNA or RNA.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides: “reference sequence”, “comparisonwindow”, “sequence identity”, “percentage of sequence identity”, and“substantial identity”. A “reference sequence” is a defined sequenceused as a basis for a sequence comparison; a reference sequence may be asubset of a larger sequence, for example, as a segment of a full-lengthcDNA or gene sequence given in a sequence listing, such as apolynucleotide sequence shown in FIG. 5 or FIG. 10, or may comprise acomplete cDNA or gene sequence. Generally, a reference sequence is atleast 20 nucleotides in length, frequently at least 25 nucleotides inlength, and often at least 50 nucleotides in length. Since twopolynucleotides may each (1) comprise a sequence (i.e., a portion of thecomplete polynucleotide sequence) that is similar between the twopolynucleotides, and (2) may further comprise a sequence that isdivergent between the two polynucleotides, sequence comparisons betweentwo (or more) polynucleotides are typically performed by comparingsequences of the two polynucleotides over a “comparison window” toidentify and compare local regions of sequence similarity. A “comparisonwindow”, as used herein, refers to a conceptual segment of at least 20contiguous nucleotide positions wherein a polynucleotide sequence may becompared to a reference sequence of at least 20 contiguous nucleotidesand wherein the portion of the polynucleotide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) of 20 percent orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by the local homology algorithm of Smith & Waterman, Appl.Math. 2:482 (1981), by the homology alignment algorithm of Needleman &Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity methodof Pearson & Lipman, Proc. Natl. Acad. Sci. (USA) 85:2444 (1988), bycomputerized implementations of these algorithms (FASTDB(Intelligenetics), BLAST (National Center for Biomedical Information) orGAP, BESTFIT, FASTA, and TFASTA (Wisconsin Genetics Software PackageRelease 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.)),or by inspection, and the best alignment (i.e., resulting in the highestpercentage of sequence similarity over the comparison window) generatedby the various methods is selected. The term “sequence identity” meansthat two polynucleotide sequences are identical (i.e., on anucleotide-by-nucleotide basis) over the window of comparison. The term“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U, or I) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity. The terms “substantial identity” as used herein denotes acharacteristic of a polynucleotide sequence, wherein the polynucleotidecomprises a sequence that has at least 70, 80 or 85 percent sequenceidentity, preferably at least 90 to 95 percent sequence identity, moreusually at least 99 percent sequence identity as compared to a referencesequence over a comparison window of at least 20 nucleotide positions,frequently over a window of at least 25-50 nucleotides, wherein thepercentage of sequence identity is calculated by comparing the referencesequence to the polynucleotide sequence which may include deletions oradditions which total 20 percent or less of the reference sequence overthe window of comparison. The reference sequence may be a subset of alarger sequence, for example, as a segment of the full-length ACT-4-h-1sequence shown in FIG. 5 or a segment of the full-length ACT-4-L-h-1sequence shown in FIG. 10.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsBLAZE (Intelligenetics) GAP or BESTFIT using default gap weights, shareat least 70 percent or 80 percent sequence identity, preferably at least90 percent sequence identity, more preferably at least 95 percentsequence identity or more (e.g., 99 percent sequence identity).Preferably, residue positions which are not identical differ byconservative amino acid substitutions. Conservative amino acidsubstitutions refer to the interchangeability of residues having similarside chains. For example, a group of amino acids having aliphatic sidechains is glycine, alanine, valine, leucine, and isoleucine; a group ofamino acids having aliphatic-hydroxyl side chains is serine andthreonine; a group of amino acids having amide-containing side chains isasparagine and glutamine; a group of amino acids having aromatic sidechains is phenylalanine, tyrosine, and tryptophan; a group of aminoacids having basic side chains is lysine, arginine, and histidine; and agroup of amino acids having sulfur-containing side chains is cysteineand methionine. Preferred conservative amino acids substitution groupsare: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, and asparagine-glutamine.

The term “substantially pure” means an object species is the predominantspecies present (i.e., on a molar basis it is more abundant than anyother individual species in the composition), and preferably asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present. Generally, a substantially purecomposition will comprise more than about 80 to 90 percent of allmacromolecular species present in the composition. Most preferably, theobject species is purified to essential homogeneity (contaminant speciescannot be detected in the composition by conventional detection methods)wherein the composition consists essentially of a single macromolecularspecies.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally-occurring.

The term “epitope” includes any protein determinant capable of specificbinding to an immunoglobulin or T-cell receptor. Epitopic determinantsusually consist of chemically active surface groupings of molecules suchas amino acids or sugar side chains and usually have specific threedimensional structural characteristics, as well as specific chargecharacteristics.

Specific binding exists when the dissociation constant for a dimericcomplex is ≦1 μM, preferably ≦100 nM and most preferably ≦1 nM.

The term “higher cognate variants” as used herein refers to a genesequence that is evolutionarily and functionally related between humansand higher mammalian species, such as primates, porcines and bovines.The term does not include gene sequences from rodents, such as rats.Thus, the cognate primate gene to the ACT-4-h-1 gene is the primate genewhich encodes an expressed protein which has the greatest degree ofsequence identity to the ACT-4-h-1 receptor protein and which exhibitsan expression pattern similar to that of the ACT-4-h-1 protein (i.e.,expressed on activated CD4⁺ cells). Similarly, the cognate primate geneto the ACT-4-L-h-1 gene is the gene whose expressed protein showsgreatest sequence identity to the ACT-4-L-h-1 ligand protein and whichexhibits a similar expression pattern (i.e., expressed on activatedB-cells).

A population of cells is substantially enriched in a selected cell typewhen that cell type constitutes at least 30, 50 or 70% of thepopulation.

The term “patient” includes human and veterinary subjects.

A test substance competes with a reference for specific binding to anantigen when an excess of the test substance substantially inhibitsbinding of the reference in a competition assay. Numerous types ofcompetition assay including radioimmunoassay and ELISA are available.See Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor(1988). Substantially inhibits means that the test substance reducesspecific binding of the reference usually by at least 10%, 25%, 50%,75%, or 90%. Test substances identified by a competition assay includethose binding to the same epitope as the reference and those binding toan adjacent epitope sufficiently proximal to that of the epitope boundby the reference antibody for steric hindrance to occur.

DETAILED DESCRIPTION I. ACT-4 Receptor Polypeptides

According to one embodiment of the invention, receptors on the surfaceof activated CD4⁺ T-cells (referred to as ACT-4 receptors) and fragmentsthereof are provided. The term ACT-4 receptor polypeptide is usedgenerically to encompass full-length proteins and fragments thereof. Theterm ACT-4 receptor is usually reserved for full-length proteins. Theamino acid sequence of the first ACT-4 receptor to be characterized[hereinafter ACT-4-h-1] is shown in FIG. 5. The suffix -h designateshuman origin and the suffix -1 indicates that ACT-4-h-1 is the firstACT-4 receptor to be characterized. The term ACT-4 receptor refers notonly to the protein having the sequence shown in FIG. 5, but also toother proteins that represent allelic, nonallelic, and higher cognatevariants of ACT-4-h-1, and natural or induced mutants of any of these.Usually, ACT-4 receptor polypeptides will also show substantial sequenceidentity with the ACT-4-h-1 sequence. Typically, an ACT-4 receptorpolypeptide will contain at least 4 and more commonly 5, 6, 7, 10 or 20,50 or more contiguous amino acids from the ACT-4-h-1 sequence. It iswell known in the art that functional domains, such as binding domainsor epitopes can be formed from as few as four amino acids residues.

ACT-4 receptor polypeptides will typically exhibit substantial aminoacid sequence identity with the amino acid sequence of ACT-4-h-1, and beencoded by nucleotide sequences that exhibit substantial sequenceidentity with the nucleotide sequence encoding ACT-4-h-1 shown in FIG.5. The nucleotides encoding ACT-4 receptor proteins will also typicallyhybridize to the ACT-4-h-1 sequence under stringent conditions. However,these nucleotides will not usually hybridize under stringent conditionsto the nucleic acid encoding OX-40 receptor, as described by Mallet etal., EMBO J. 9:1063-68 (1990) (hereby incorporated by reference for allpurposes) (See particularly FIG. 2A of the Mallet et al. reference).Stringent conditions are sequence dependent and will be different indifferent circumstances. Generally, stringent conditions are selected tobe about 5° C. lower than the thermal melting point (Tm) for thespecific sequence at a defined ionic strength and pH. The Tm is thetemperature (under defined ionic strength and Ph) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Typically,stringent conditions will be those in which the salt concentration is atleast about 0.02 molar at Ph 7 and the temperature is at least about 60°C. As other factors may significantly affect the stringency ofhybridization, including, among others, base composition and size of thecomplementary strands, the presence of organic solvents and the extentof base mismatching, the combination of parameters is more importantthan the absolute measure of any one.

Usually, ACT-4 receptor polypeptides will share at least one antigenicdeterminant in common with ACT-4-h-1 but will not be specificallyreactive with antibodies against the rat OX-40 polypeptide. Theexistence of a common antigenic determinant is evidenced bycross-reactivity of the variant protein with any antibody preparedagainst ACT-4-h-1 (see Section IV). Cross-reactivity is often testedusing polyclonal sera against ACT-4-h-1, but can also be tested usingone or more monoclonal antibodies against ACT-4-h-1, such as theantibody designated L106.

Often ACT-4 receptor polypeptides will contain modified polypeptidebackbones. Modifications include chemical derivatizations ofpolypeptides, such as acetylations, carboxylations and the like. Theyalso include glycosylation modifications (N- and O-linked) andprocessing variants of a typical polypeptide. These processing stepsspecifically include enzymatic modifications, such as ubiquitinizationand phosphorylation. See, e.g., Hershko & Ciechanover, Ann. Rev. Bioch.51:335-364 (1982). The ACT-4-h-1 protein, for example, is heavilymodified in that the observed molecular weight is about 50 kDa, whereasthe predicted molecular weight based on amino acid sequence is only 27kDa. Two putative glycosylation sites have been identified in itsextracellular domain.

ACT-4 receptors likely share some or all of the topological featuresfound for ACT-4-h-1. The amino acid sequence for ACT-4-h-1 contains a 22or 24 amino acid putative N-terminal signal sequence. The 24 amino acidsequence is more probably based on the criteria of von Heijne, NucleicAcids Res. 14:4683-4690 (1986) (incorporated by reference for allpurposes). The ACT-4-h-1 receptor contains a single additionalhydrophobic stretch of 27 amino acids spanning residues 213-240. Thehydrophobic stretch probably corresponds to a transmembrane domain andits existence is consistent with ACT-4-h-1 being a type I integralmembrane protein (i.e., having a single transmembrane domain with theN-terminal domain comprising the extracellular region and the C-terminuscomprising the intracellular region). The 189 or 191 amino acids(depending on the exact location of the signal cleavage site) ofACT-4-h-1 amino-proximal to the transmembrane segment are designated theextracellular domain, while the 37 amino acids carboxy-proximal to thetransmembrane segment are designated the intracellular domain. From theamino-terminus, the extracellular domain has an NH₂-terminal hydrophobicputative signal sequence, and three intrachain loops formed by disulfidebonding between paired cysteine residues.

The topological arrangement of ACT-4 receptor polypeptides is similar tothat of other members of the nerve growth factor receptor family,particularly to the rat OX-40 receptor. However, the other members showsome divergence in the number of extracellular disulfide loops andglycosylation sites and in the size of the intracellular domain. SeeMallet & Barclay, supra.

Although not all of the domains discussed above are necessarily presentin all ACT-4 receptor polypeptides, an extracellular domain is expectedto be present in most. Indeed, in some ACT-4 receptor polypeptides, itis possible that only an extracellular domain is present, and thenatural state of such proteins is not as cell-surface bound proteins,but as soluble proteins, for example, dispersed in an extracellular bodyfluid. The existence of soluble variant forms has been observed forother cell surface receptors, including one member of the nerve growthfactor receptor family, SFV-T2. See Mallet & Barclay, supra.

Besides substantially full-length polypeptides, the present inventionprovides for biologically active fragments of the polypeptides.Significant biological activities include receptor binding, antibodybinding (e.g., the fragment competes with an intact ACT-4 receptor forspecific binding to an antibody), immunogenicity (i.e., possession ofepitopes that stimulate B or T cell responses against the fragment), andagonism or antagonism of the binding of an ACT-4 receptor polypeptide toits ligands. A segment of an ACT-4 receptor protein or a domain thereofwill ordinarily comprise at least about 5, 7, 9, 11, 13, 16, 20, 40, or100 contiguous amino acids.

Segments of ACT-4 receptor polypeptides are often terminated nearboundaries of functional or structural domains. Such segments consistessentially of the amino acids responsible for a particular functionalor structural property. Structural and functional domains are identifiedby comparison of nucleotide and/or amino acid sequence data such as isshown in FIG. 5 to public or proprietary sequence databases. Preferably,computerized comparison methods are used to identify sequence motifs orpredicted protein conformation domains that occur in other proteins ofknown structure and/or function. Structural domains include anintracellular domain, transmembrane domain, and extracellular domain,which is in turn contains three disulfide-bonded loops. Functionaldomains include an extracellular binding domain through which the ACT-4receptor polypeptide interacts with external soluble molecules or othercell-bound ligands and an intracellular signal-transducing domain.

Some fragments will contain only extracellular domains, such as one ormore disulfide-bonded loops. Such fragments will often retain thebinding specificity of an intact ACT-4 receptor polypeptide, but will besoluble rather than membrane bound. Such fragments are useful ascompetitive inhibitors of ACT-4 receptor binding.

ACT-4 receptors are further identified by their status as members of thenerve growth factor receptor family. The amino acid sequence ofACT-4-h-1 is at least 20% identical to NGF-R, TNF-R, CD40, 4-1BB, andfas/APO1. ACT-4-h-1 exhibits 62% amino acid sequence identity with therat OX-40 gene, which is also characterized by selective expression onactivated CD4⁺ cells.

ACT-4 receptors are also identified by a characteristic cellulardistribution. Most notably, ACT-4 receptors are usually easily detectedon activated CD4⁺ T cells (percent cells expressing usually greater thanabout 25 or 50% and often about 80%; mean channel fluorescence usuallygreater than about 10 and often about 20-25, on a Coulter Profile FlowCytometer after immunofluorescence staining). ACT-4 receptors areusually substantially absent on resting T-cells, B-cells (unlessactivated with PMA), NK cells, and monocytes (unless activated withPMA). Substantially absent means that the percentage of cells expressingACT-4 is usually less than about 5%, and more usually less than about2%, and that the mean channel is usually less than about 4, and moreusually less than about 2, measured on a Coulter Profile Flow Cytometer,after immunofluorescence staining of the cells. (See Example 2) ACT-4receptors are usually expressed at low levels on, activated CD8⁺ cells(percent cells expressing about 4-10%; mean channel fluorescence about2-4 on a Coulter Profile Flow Cytometer after immunofluorescencestaining). The low level of expression observed on CD8⁺ cells suggeststhat expression is confined to a subpopulation of CD8⁺ cells. Theexpression of ACT-4 receptors on the surface of activated CD4⁺ cells hasbeen observed for several different mechanisms of activation, includingalloantigenic, tetanus toxoid or mitogenic (e.g., PHA) stimuli.Expression peaks after about 7 days of allogantigenic or tetanus toxoidstimulation and after about three days of PHA stimulation. These dataindicate that ACT-4 receptors should be classified as early activationantigens that are substantially absent on resting cells. The observationthat ACT-4 receptors are preferentially expressed on activated CD4⁺cells and are expressed to a much lesser extent on activated CD8⁺ cells,but are substantially absent on most or all other subtypes of lymphoidcells (except in response to highly nonphysiological stimuli such asPMA) contrasts with the cell type specificity of other activationantigens found on human leukocytes.

The expression of ACT-4 receptors on the surface of activated CD4⁺ Tcells suggests that the receptor has a role in activation of thesecells. Such a role is consistent with that of some other members of thenerve growth factor receptor family. For example, CD40 stimulates theG1-S phase transition in B lymphocytes, and nerve growth factor receptortransduces a signal from the cytokine nerve growth factor, which resultsin neuronal differentiation and survival (Bards, Neuron 2:1525-1534(1989)) (incorporated by reference for all purposes). However, otherroles for ACT-4 receptors can also be envisaged, for example,interaction with other lymphoid cell types. The existence of such rolesis consistent with the diverse functions of other nerve growth factorreceptor family members, such as tumor necrosis factor, whoseinteraction with tumor necrosis factor receptor can result ininflammation or tumor cell death.

Fragments or analogs comprising substantially one or more functionaldomain (e.g., an extracellular domain) of ACT-4 receptors can be fusedto heterologous polypeptide sequences, such that the resultant fusionprotein exhibits the functional property(ies) conferred by the ACT-4receptor fragment and/or the fusion partner. The orientation of theACT-4 receptor fragment relative to the fusion partner will depend onexperimental considerations such as ease of construction, stability toproteolysis, thermal stability, immunological reactivity, amino- orcarboxyl-terminal residue modification, and so forth. Potential fusionpartners include chromogenic enzymes such as β-galactosidase, protein Aor G, a FLAG protein such as described by Blanar & Rutter, Science256:1014-1018 (1992), toxins (e.g., diphtheria toxin, Psuedonomasectotoxin A, ricin toxin or phospholipase C) and immunoglobulincomponents.

Recombinant globulins (Rg) formed by fusion of ACT-4 receptor fragmentsand immunoglobulin components often have most or all of thephysiological properties associated with the constant region of theparticular immunoglobulin class used. For example, the recombinantglobulins may be capable of fixing complement, mediating antibodydependent cell toxicity, stimulating B cells, or traversing blood vesselwalls and entering the interstitial space. The recombinant globulins areusually formed by fusing the C-terminus of an ACT-4 receptorextracellular domain to the N-terminus of the constant region domain ofa heavy chain immunoglobulin, thereby simulating the conformation of anauthentic immunoglobulin chain. The immunoglobulin chain is preferablyof human origin, particularly if the recombinant globulin is intendedfor therapeutic use. Recombinant globulins are usually soluble and havea number of advantageous properties relative to unmodified ACT-4receptors. These properties include prolonged serum half-life, thecapacity to lyse target cells for which an ACT-4 receptor has affinity,by effector functions, and the capacity to bind molecules such asprotein A and G, which can be used to immobilize the recombinantglobulin in binding analyses.

II. Ligands to ACT-4

The invention also provides ligands that specifically bind to an ACT-4receptor polypeptide and that are capable of forming a complex with sucha polypeptide, at least in part, by noncovalent binding. The termACT-4-L ligand polypeptide is used generically to encompass full-lengthproteins and fragments thereof. This term does not usually includeantibodies to ACT-4 receptor polypeptides. The term ACT-4 ligand isusually used to refer to a full-length protein. Ligands can benaturally-occurring or synthetic molecules, and can be in soluble formor anchored to the surface of a cell. Multiple different ligands maybind the same ACT-4 receptor. Conversely, one ligand may bind to morethan one ACT-4 receptor. Usually, binding of a ligand to an ACT-4receptor will initiate a signal that alters the physical and/orfunctional phenotype of a cell bearing the ACT-4 receptor and/or a cellbearing the ACT-4 ligand. Antibodies against either ACT-4 or its ligandscan have the capacity to block or stimulate signal transduction. Itwill, of course, be recognized that the designation of ACT-4 as areceptor and its specific binding partner(s) as ligand(s) is somewhatarbitrary and might, in some circumstances, be reversed.

Source materials for supplying ACT-4-L ligand polypeptides areidentified by screening different cell types, particularly lymphoid andhematopoietic cells, bodily fluids and tissue extracts, with labelledACT-4 receptor polypeptides, preferably in aqueous-soluble form, as aprobe. Activated B cells or B cell lines may be suitable (see Example8). HTLV-I infected T-cells are also suitable. Often, the ACT-4 receptoror a binding fragment thereof is fused or otherwise linked to a secondprotein for purposes of screening. Particularly suitable are recombinantglobulins formed by fusing the extracellular portion of ACT-4-h-1 to theconstant region of an immunoglobulin heavy chain.

ACT-4-L ligand polypeptides are purified from cells or other biologicalmaterials identified by this screening method using techniques ofclassical protein chemistry. Such techniques include selectiveprecipitation with such substances as ammonium sulfate, columnchromatography, immunopurification methods, and others. See, e.g., R.Scopes, Protein Purification: Principles and Practice (Springer-Verlag,NY, 1982) (incorporated by reference for all purposes). Usually,purification procedures will include an affinity chromatography step inwhich an ACT-4 receptor polypeptide or a binding fragment thereof isused as the immobilized reagent. ACT-4-constant regions can beconveniently immobilized by binding of the constant region moiety toprotein A or G. ACT-4-L ligand polypeptides can also be purified usinganti-idiotypic antibodies to ACT-4 receptors as the affinity reagent.

To determine the amino acid sequence or to obtain polypeptide fragmentsof a ligand, the ligand can be digested with trypsin. Peptide fragmentscan be separated by reverse-phase high-performance liquid chromatography(HPLC), and analyzed by gas-phase sequencing. Other sequencing methodsknown in the art may also be used. The sequence data can be used todesign degenerate probes for isolation of cDNA or genomic clonesencoding ACT-4-L ligand polypeptides.

Alternatively, cDNA clones encoding ACT-4-L ligand polypeptides can beobtained by expression cloning. In this approach, a cDNA library isprepared from cells expressing an ACT-4-L ligand polypeptides(identified as discussed, supra). The library is expressed inappropriate cells (e.g., COS-7), and clones bearing the ACT-4-L ligandpolypeptide are identified by screening with labelled ACT-4 or bindingfragment thereof, optionally fused to a constant domain of animmunoglobulin heavy chain.

The cDNA sequence and predicted amino acid sequence of the first ligandto an ACT-4 receptor polypeptide to be characterized are shown in FIG.10. This ligand is designated ACT-4-L-h-1 with the suffix h designatinghuman origin, and the suffix 1 indicating that this is the first ligandto be characterized. The coding portion of the cDNA sequence ofACT-4-L-h-1 is identical or nearly identical to that of a polypeptidetermed gp34 or TA34. See Miura et al., Mol. Cell. Biol. 11:1313-1325(1991) (incorporated by reference in its entirety for all purposes). Theinvention also includes ligands representing allelic, nonallelic, spliceand higher cognate variants of ACT-4-L-h-1, and natural or inducedmutants of any of these. Such variants will typically show substantialsequence identity with the ACT-4-L-h-1 sequence, and contain at least 4and more commonly 5, 6, 7, 10 or 20, 50 or more contiguous amino acidsfrom the ACT-4-L-h-1 sequence. Such variants will also typically beencoded by nucleotide sequences that exhibit substantial sequenceidentity with the nucleotide sequence encoding ACT-4-L-h-1 shown in FIG.10. The nucleotides encoding such variants will also typically hybridizeto an ACT-4-L-h-1 DNA sequence under stringent conditions. However, somesuch nucleotides will not hybridize under stringent conditions to DNAsequences encoding lower cognate variants (e.g. rat) of ACT-4-L-h-1.Many variants of ACT-4-L-h-1 will share at least one antigenicdeterminant with an ACT-4-L-h-1 ligand polypeptide as evidenced bycrossreactivity with monoclonal or polyclonal antibodies against thesame. However, some such variants will not crossreact with sera againstlower cognate variants of ACT-4-L-h-1.

Although many ACT-4-L ligand polypeptides will show similarity toACT-4-L-h-1 in at least one of the respects discussed above, it isentirely possible that other families of ligands exists to the ACT-4receptor that show nothing in common with ACT-4-L-h-1 except for thecapacity to exist, in part, as type II integral membrane cell surfaceproteins. Superfamily ligands also exists as soluble proteins suggestingthat such forms exist for ACT-4-L ligand polypeptides. The C-terminalextracellular domain of ACT-4-L-h-1 shows some sequence similarity withvarious dehydrogenases. Thus, some ACT-4-L ligand polypeptide maypossess a dehydrogenase activity, which may play a role in intercellularsignalling.

Besides substantially full-length polypeptides, the present inventionprovides for biologically active fragments of full-length ACT-4-L ligandpolypeptides. Significant biological activities include binding to anACT-4 receptor such as ACT-4-h-1, binding to a second ACT-4-L ligandpolypeptide, antibody binding (e.g., the fragment competes with anintact ACT-4-L-h-1 ligand polypeptide for specific binding to anantibody), immunogenicity (i.e., possession of epitopes that stimulate Bor T cell responses against the fragment), and agonism or antagonism ofthe binding of a second ACT-4-L ligand polypeptide to an ACT-4 receptorpolypeptide, such as ACT-4-h-1. A segment of a full-length ACT-4-Lligand polypeptide will ordinarily comprise at least 5 contiguous aminoacids, but not more than 160 contiguous amino acids from the amino acidsequence shown in FIG. 10. Often segments contain about 10, 20, 50, 75,100 or 133 amino acids, and not more than 150 contiguous amino acids,from the sequence shown in FIG. 10.

Some fragments will contain only extracellular domains. Such fragmentscontain the full-length domains of naturally occurring ACT-4-L ligandpolypeptides. Other fragments contain components thereof. Such fragmentswill often retain the binding specificity of an intact ACT-4-L ligandpolypeptide, but will be soluble rather than membrane bound. Suchfragments are useful as competitive inhibitors of ACT-4-L ligandpolypeptide binding to a receptor.

Fragments of full-length ACT-4-L ligand polypeptides are oftenterminated at one or both of their ends near (i.e., within about 5, 10or 20 aa of) the boundaries of functional or structural domains.Fragments terminated at both ends by structural or functional boundariesconsist essentially of a particular segment (or domain) of ACT-4-L aminoacids responsible for a functional or structural property. Structuraland functional domains are identified by comparison of nucleotide and/oramino acid sequence data such as is shown in FIG. 10 to public orproprietary sequence databases. Preferably, computerized comparisonmethods are used to identify sequence motifs or predicted proteinconformation domains that occur in other proteins of known structureand/or function. Binding domains can be identified by epitope mapping.See Section VI, infra. Structural domains include an intracellulardomain, transmembrane domain, and extracellular domain. Functionaldomains include an extracellular binding domain through which ACT-4-Lligand polypeptides interact with external soluble molecules orcell-bound receptors and an intracellular signal-transducing domain.

Expression of ACT-4-L-h-1 and related ligands is dependent on cell typeand activation status. See Example 8. Most notably, ACT-4-L-h-1 iseasily detected on some PMA/ionomycin-activated B cell lines.ACT-4-L-h-1 is substantially absent on fresh resting B cells.ACT-4-L-h-1 is also expressed on HLTV-1-infected T-cells and may beinfect on other T-cell types in some circumstances. See Example 8.

The affinity of an ACT-4-L ligand (expressed on activated B cells) foran ACT-4 receptor (expressed Predominantly on activated CD4⁺ cells)suggests that the interaction between ligand and receptor may have arole in activation/differentiation of CD4⁺ T-cells and/or B cells. BothCD4⁺ T-cell and B-cell activation are known to be multistep processesrequiring antigen-specific and nonspecific stimuli. The interactionbetween ACT-4 and ACT-4-L would likely constitute a nonantigen-specificstimulus effective on either or both of the respective cells bearingthese antigens. The stimulus might be direct when as, for example, theligand-receptor binding triggers an enzymic activity in theintracellular domain in ligand and/or receptor, which activity in turninitiates a cascade of metabolic events in one or both of the respectivecells. Alternatively, the stimulus might be indirect; for example, theinteraction between ACT-4 and ACT-4-L might increase the avidity ofcellular interactions between other ligand-receptor pairs or controlleukocyte localization and migration. Interaction of ACT-4 and ACT-4-Lmay act in conjunction with binding of other T_(H)-B cellreceptor/ligand pairs such as CD2/LFA-3 (Moingeon et al., Nature 339:314(1988)), CD4/MHC class II (Doyle & Stominger, Nature 330:256-259(1987)), LFA-1/ICAM-1/ICAM-2 (Makgoba et al., Nature 331:86-88 (1988)and Staunton et al., Nature 339:61-64 (1989)) and B7/CD28 (Linsley etal., J. Exp. Med. 173:721-730 (1991)). ACT-4/ACT-4-L interactions mayalso be enhanced or diminished by binding of soluble molecules to CD4⁺T-cells and/or B cells. Likely soluble molecules are cytokinesincluding, e.g., interleukins IL-1 through IL-13, tumor necrosis factorsα & β, interferons α, β and γ, tumor growth factor Beta (TGF-β), colonystimulating factor (CSF) and granulocyte monocyte colony stimulatingfactor (GM-CSF).

Expression of ACT-4-L ligand on certain subtypes of T-cells in somecircumstances may confer additional or alternate roles for ACT-4/ACT-4-Linteractions. For instance, efficiency of infection of T-cells by humanimmunodeficiency virus (HIV) or other viruses and pathogens may beaffected by ACT-4 or ACT-4-L or by interactions between the two. Inaddition it is possible that ACT-4 and ACT-4-L could be expressed by thesame cells (e.g., activated CD4⁺ T-cells). In these circumstances,interactions between receptor and ligand may affect the growth andactivation state of their respective cells.

Fragments or analogs comprising substantially one or more functionaldomain (e.g., an extracellular domain) of ACT-4-L ligand polypeptidescan be fused or otherwise linked to heterologous polypeptide sequences,such that the resultant fusion protein exhibits the functionalproperty(ies) conferred by the ACT-4-L ligand polypeptide and/or thefusion partner. Suitable fusion partners are as discussed in Section I,supra.

The ACT-4-L ligand polypeptides can be used to affinity purifyrespective ACT-4 receptors. ACT-4-L ligand polypeptides are also usefulas agonists or antagonists of a second ACT-4-L ligand or an ACT-4receptor, and can be used in the therapeutic methods discussed inSection VII, infra. ACT-4 ligand polypeptides are also useful inscreening assays for identifying agonists and antagonists of ACT-4and/or ACT-4-L.

III. Methods of Producing Polypeptides

A. Recombinant Technologies

The nucleotide and amino acid sequences of ACT-4-h-1 shown in FIG. 5,and corresponding sequences for other ACT-4 receptor variants allowproduction of polypeptides of full-length ACT-4 receptor polypeptidessequences and fragments thereof. Similarly, the amino acid sequence ofACT-4-L-h-1 and corresponding sequences for other ACT-4-L ligandpolypeptide variants allow production of full-length and fragment ligandpolypeptides. Such polypeptides may be produced in prokaryotic oreukaryotic host cells by expression of polynucleotides encoding ACT-4 orACT-4-L, or fragments and analogs of either of these. The cloned DNAsequences are expressed in hosts after the sequences have been operablylinked to (i.e., positioned to ensure the functioning of) an expressioncontrol sequence in an expression vector. Expression vectors aretypically replicable in the host organisms either as episomes or as anintegral part of the host chromosomal DNA. Commonly, expression vectorswill contain selection markers, e.g., tetracycline resistance orhygromycin resistance, to permit detection and/or selection of thosecells transformed with the desired DNA sequences (see, e.g., U.S. Pat.No. 4,704,362).

E. coli is one prokaryotic host useful for cloning the DNA sequences ofthe present invention. Other microbial hosts suitable for use includebacilli, such as Bacillus subtilis, and other Enterobacteriaceae, suchas Salmonella, Serratia, and various Pseudomonas species. In theseprokaryotic hosts, one can also make expression vectors, which willtypically contain expression control sequences compatible with the hostcell (e.g., an origin of replication). In addition, any number of avariety of well-known promoters will be present, such as the lactosepromoter system, a tryptophan (trp) promoter system, a beta-lactamasepromoter system, or a promoter system from phage lambda. The promoterswill typically control expression, optionally with an operator sequence,and have ribosome binding site sequences and the like, for initiatingand completing transcription and translation.

Other microbes, such as yeast, may also be used for expression.Saccharomyces is a preferred host, with suitable vectors havingexpression control sequences, such as promoters, including3-phosphoglycerate kinase or other glycolytic enzymes, and an origin ofreplication, termination sequences and the like as desired. Insect cells(e.g., SF9) with appropriate vectors, usually derived from baculovirus,are also suitable for expressing ACT-4 receptor or ligand polypeptides.See Luckow, et al. Bio/Technology 6:47-55 (1988) (incorporated byreference for all purposes).

Higher eukaryotic mammalian tissue cell culture may also be used toexpress and produce the polypeptides of the present invention (seeWinnacker, From Genes to Clones (VCH Publishers, NY, N.Y., 1987))(incorporated by reference for all purposes). Eukaryotic cells areactually preferred, because a number of suitable host cell lines capableof secreting and authentically modifying human proteins have beendeveloped in the art, and include the CHO cell lines, various COS celllines, HeLa cells, myeloma cell lines, Jurkat cells, etc. Expressionvectors for these cells can include expression control sequences, suchas an origin of replication, a promoter (e.g., a HSV tk promoter or pgk(phosphoglycerate kinase) promoter), an enhancer (Queen et al., Immunol.Rev. 89:49 (1986)), and necessary processing information sites, such asribosome binding sites, RNA splice sites, polyadenylation sites (e.g.,an SV40 large T Ag poly A addition site), and transcriptional terminatorsequences. Preferred expression control sequences are promoters derivedfrom immunoglobulin genes, SV40, adenovirus, bovine papillomavirus, andthe like. The vectors containing the DNA segments of interest (e.g.,polypeptides encoding an ACT-4 receptor) can be transferred into thehost cell by well-known methods, which vary depending on the type ofcellular host. For example, CaCl₂ transfection is commonly utilized forprokaryotic cells, whereas CaPO₄ treatment or electroporation may beused for other cellular hosts. Vectors may exist as episome orintegrated into the host chromosome.

B. Naturally Occurring ACT-4 or ACT-4-L Polypeptides

Natural ACT-4 receptor polypeptides are isolated by conventionaltechniques such as affinity chromatography. For example, polyclonal ormonoclonal antibodies are raised against previously-purified ACT-4-h-1and attached to a suitable affinity column by well known techniques.See, e.g., Hudson & Hay, Practical Immunology (Blackwell ScientificPublications, Oxford, UK, 1980), Chapter 8 (incorporated by referencefor all purposes). For example, anti-ACT-4-h-1 can be immobilized to aprotein-A sepharose column via crosslinking of the F_(c) domain with ahomobifunctional crosslinking agent, such as dimethyl pimelimidate. Cellextracts are then passed through the column, and ACT-4 receptor proteinspecifically bound by the column, eluted with, for example, 0.5 Mpyrogenic acid, pH 2.5. Usually, an intact form of ACT-4 receptor isobtained by such isolation techniques. Peptide fragments are generatedfrom intact ACT-4 receptors by chemical (e.g., cyanogen bromide) orenzymatic cleavage (e.g., V8 protease or trypsin) of the intactmolecule.

Naturally occurring ACT-4-L ligand polypeptides can be purified using ananalogous approach except that the affinity reagent is an antibodyspecific for ACT-4-L-h-1.

C. Other Methods

Alternatively, ACT-4 or ACT-4-L polypeptides can be synthesized bychemical methods or produced by in vitro translation systems using apolynucleotide template to direct translation. Methods for chemicalsynthesis of polypeptides and in vitro translation are well known in theart, and are described further by Berger & Kimmel, Methods inEnzymology, Volume 152, Guide to Molecular Cloning Techniques AcademicPress, Inc., San Diego, Calif., 1987).

IV. Nucleic Acids

A. Cloning ACT-4 or ACT-4-L Nucleic Acids

Example 5 presents nucleic acid sequence data for a cDNA clone of anACT-4 receptor designated ACT-4-h-1. The sequence includes both atranslated region and 3′ and 5′ flanking regions. This sequence data canbe used to design probes with which to isolate other ACT-4 receptorgenes. These genes include the human genomic gene encoding ACT-4-h-1,and cDNAs and genomic clones from higher mammalian species, and allelicand nonallelic variants, and natural and induced mutants of all of thesegenes. Specifically, all nucleic acid fragments encoding all ACT-4receptor polypeptides'disclosed in this application are provided.Genomic libraries of many species are commercially available (e.g.,Clontech, Palo Alto, Calif.), or can be isolated de novo by conventionalprocedures. cDNA libraries are best prepared from activated CD4⁺ cells,which express ACT-4-h-1 in large amounts.

The probes used for isolating clones typically comprise a sequence ofabout at least 24 contiguous nucleotides (or their complement) of thecDNA sequence shown in FIG. 5. For example, a full-length polynucleotidecorresponding to the sequence shown in FIG. 5 can be labeled and used asa hybridization probe to isolate genomic clones from a human genomicclone library in e.g., λEMBL4 or λGEM11 (Promega Corporation, Madison,Wis.); typical hybridization conditions for screening plaque lifts(Benton & Davis, Science 196:180 (1978)) can be: 50% formamide, 5×SSC orSSPE, 1-5×Denhardt's solution, 0.1-1% SDS, 100-200 μg shearedheterologous DNA or tRNA, 0-10% dextran sulfate, 1×10⁵ to 1×10⁷ cpm/mlof denatured probe with a specific activity of about 1×10⁸ cpm/μg, andincubation at 42° C. for about 6-36 hours. Prehybridization conditionsare essentially identical except that probe is not included andincubation time is typically reduced. Washing conditions are typically1-3×SSC, 0.1-1% SDS, 50-70° C. with change of wash solution at about5-30 minutes. Hybridization and washing conditions are typically lessstringent for isolation of higher cognate or nonallelic variants thanfor e.g., the human genomic clone of ACT-4-h-1.

Alternatively, probes can be used to clone ACT-4 receptor genes bymethods employing the polymerase chain reaction (PCR). Methods for PCRamplification are described in e.g., PCR Technology: Principles andApplications for DNA Amplification (ed. H. A. Erlich, Freeman Press, NY,N.Y., 1992); PCR Protocols: A Guide to Methods and Applications (eds.Innis, et al., Academic Press, San Diego, Calif., 1990); Mattila et al.,Nucleic Acids Res. 19:4967 (1991); Eckert, K. A. and Kunkel, T. A., PCRMethods and Applications 1:17 (1991); PCR (eds. McPherson et al., IRLPress, Oxford); and U.S. Pat. No. 4,683,202 (each of which isincorporated by reference for all purposes).

Alternatively, synthetic polynucleotide sequences corresponding to allor part of the sequences shown in FIG. 5 may be constructed by chemicalsynthesis of oligonucleotides.

Nucleotide substitutions, deletions, and additions can be incorporatedinto the polynucleotides of the invention. Nucleotide sequence variationmay result from degeneracy of the genetic code, from sequencepolymorphisms of various ACT-4 receptor alleles, minor sequencingerrors, or may be introduced by random mutagenesis of the encodingnucleic acids using irradiation or exposure to EMS, or by changesengineered by site-specific mutagenesis or other techniques of modernmolecular biology. See Sambrook et al., Molecular Cloning: A LaboratoryManual (C.S.H.P. Press, NY 2d ed., 1989) (incorporated by reference forall purposes). For nucleotide sequence that are capable of beingtranscribed and translated to produce a functional polypeptide,degeneracy of the genetic code results in a number of nucleotidesequences that encode the same polypeptide. The invention includes allsuch sequences. Generally, nucleotide substitutions, deletions, andadditions should not substantially disrupt the ability of an ACT-4receptor polynucleotide to hybridize to the sequence of ACT-4-h-1 shownin FIG. 5 under stringent conditions. Typically, ACT-4 receptorpolynucleotides comprise at least 25 consecutive nucleotides which aresubstantially identical to a naturally-occurring ACT-4 receptor sequence(e.g., FIG. 5), more usually ACT-4 receptor polynucleotides comprise atleast 50 to 100 consecutive nucleotides, which are substantiallyidentical to a naturally-occurring ACT-4 receptor sequence.

ACT-4 receptor polynucleotides can be short oligonucleotides (e.g.,about 10, 15, 25, 50 or 100 contiguous bases from the ACT-h-1 sequenceshown in FIG. 5), such as for use as hybridization probes and PCR (orLCR) primers. ACT-4 receptor polynucleotide sequences can also comprisepart of a larger polynucleotide that includes sequences that facilitatetranscription (expression sequences) and translation of the codingsequences, such that the encoded polypeptide product is produced.Construction of such polynucleotides is well known in the art and isdescribed further in Sambrook et al., supra (C.S.H.P. Press, NY 2d ed.1989). The ACT-4 receptor polynucleotide can be fused in frame withanother polynucleotide sequence encoding a different protein (e.g.,glutathione S-transferase, β-galactosidase or an immunoglobulin F_(c)domain) for encoding expression of a fusion protein (see, e.g., Byrn etal., Nature, 344:667-670 (1990)) (incorporated by reference for allpurposes).

Nucleic acids encoding ACT-4-L genes can be produced by an analogousapproach, using the ACT-4-L-h-1 cDNA sequence shown in FIG. 10 as astarting material for probe design. ACT-4-L genes include the humangenomic gene encoding ACT-4-L-h-1, allelic and nonallelic variants,higher cognate variants, and natural and induced mutants of all of thesegenes. Specifically, all nucleic acid fragments (genomic, cDNA orsynthetic) encoding all ACT-4-L polypeptides disclosed in thisapplication are provided. In nucleic acid, fragments having mutations ofnaturally occurring sequences, generally the mutation will notsubstantially disrupt the ability of an ACT-4-L polynucleotide tohybridize to the nucleotide sequence of ACT-4-L-h-1 under stringentconditions. ACT-4-L ligand polynucleotides can be short oligonucleotides(e.g., about 10, 15, 25, 50 or 100 contiguous bases from the ACT-4-L-h-1sequence shown in FIG. 10), such as for use as hybridization probes andPCR (or LCR) primers. ACT-4-L ligand polynucleotide sequences can alsocomprise part of a larger polynucleotide as discussed in connection withACT-4 polynucleotide sequences.

V. Antibodies and Hybridomas

In another embodiment of the invention, antibodies against ACT-4 andACT-4-L polypeptides are provided.

A. General Characteristics of Antibodies

Antibodies or immunoglobulins are typically composed of four covalentlybound peptide chains. For example, an IgG antibody has two light chainsand two heavy chains. Each light chain is covalently bound to a heavychain. In turn each heavy chain is covalently linked to the other toform a “Y” configuration, also known as an immunoglobulin conformation.Fragments of these molecules, or even heavy or light chains alone, maybind antigen. Antibodies, fragments of antibodies, and individual chainsare also referred to herein as immunoglobulins.

A normal antibody heavy or light chain has an N-terminal (NH₂) variable(V) region, and a C-terminal (—COOH) constant (C) region. The heavychain variable region is referred to as V_(H) (including, for example,V_(γ)), and the light chain variable region is referred to as V_(L)(including V_(κ) or V_(λ)). The variable region is the part of themolecule that binds to the antibody's cognate antigen, while the Fcregion (the second and third domains of the C region) determines theantibody's effector function (e.g., complement fixation, opsonization).Full-length immunoglobulin or antibody “light chains” (generally about25 kDa, about 214 amino acids) are encoded by a variable region gene atthe N-terminus (generally about 110 amino acids) and a κ (kappa) or λ(lambda) constant region gene at the COOH-terminus. Full-lengthimmunoglobulin or antibody “heavy chains” (generally about 50 Kd, about446 amino acids), are similarly encoded by a variable region gene(generally encoding about 116 amino acids) and one of the constantregion genes, e.g., gamma (encoding about 330 amino acids). Typically,the “V_(L)” will include the portion of the light chain encoded by theV_(L) and/or J_(L) (J or joining region) gene segments, and the “V_(H)”will include the portion of the heavy chain encoded by the V_(H), and/orD_(H) (D or diversity region) and J_(H) gene segments. See, generally,Roitt et al., Immunology (2d ed. 1989), Chapter 6 and Paul, FundamentalImmunology (Raven Press, 2d ed., 1989) (each of which is incorporated byreference for all purposes).

An immunoglobulin light or heavy chain variable region consists of a“framework” region interrupted by three hypervariable regions, alsocalled complementarity-determining regions or CDRs. The extent of theframework region and CDRs have been defined (see Kabat et al. (1987),“Sequences of Proteins of Immunological Interest,” U.S. Department ofHealth and Human Services; Chothia et al., J. Mol. Biol. 196:901-917(1987) (each of which is incorporated by reference for all purposes).The sequences of the framework regions of different light or heavychains are relatively conserved within a species. The framework regionof an antibody, that is the combined framework regions of theconstituent light and heavy chains, serves to position and align theCDRs in three dimensional space. The CDRs are primarily responsible forbinding to an epitope of an antigen. The CDRs are typically referred toas CDR1, CDR2, and CDR3, numbered sequentially starting from theN-terminus.

The constant region of the heavy chain molecule, also known as C_(H),determines the isotype of the antibody. Antibodies are referred to asIgM, IgD, IgG, IgA, and IgE depending on the heavy chain isotype. Theisotypes are encoded in the mu (μ), delta (Δ), gamma (γ), alpha (α), andepsilon (ε) segments of the heavy chain constant region, respectively.In addition, there are a number of γ subtypes. There are two types oflight chains, κ and λ. The determinants of these subtypes typicallyreside in the constant region of the light chain, also referred to asthe C_(L) in general, and C_(κ) or C_(λ) in particular.

The heavy chain isotypes determine different effector functions of theantibody, such as opsonization or complement fixation. In addition, theheavy chain isotype determines the secreted form of the antibody.Secreted IgG, IgD, and IgE isotypes are typically found in single unitor monomeric form. Secreted IgM isotype is found in pentameric form;secreted IgA can be found in both monomeric and dimeric form.

B. Production of Antibodies

Antibodies which bind either an ACT-4 receptor, an ACT-4-L ligand, orbinding fragments of either, can be produced by a variety of means. Theproduction of non-human monoclonal antibodies, e.g., murine, rat and soforth, is well known and may be accomplished by, for example, immunizingthe animal with a preparation containing an ACT-4 receptor or itsligands, or an immunogenic fragment of either of these. Particularly,useful as immunogens are cells stably transfected with a recombinantACT-4 or ACT-4-L gene and expressing an ACT-4 receptor or ligand theretoon their cell surface. Antibody-producing cells obtained from theimmunized animals are immortalized and screened for the production of anantibody which binds to ACT-4 receptors or their ligands. See Harlow &Lane, Antibodies, A Laboratory Manual (C.S.H.P. NY, 1988) (incorporatedby reference for all purposes). A number of murine antibodies to aprotein having a substantially similar or identical primary amino acidsequence to ACT-4-L-h-1 have been discussed by Tozawa et al., Int. J.Cancer 41:231-238 (1988); Tanaka et al., Int. J. Cancer 36:549-555(1985) (incorporated by reference in their entirety for all purposes).

Several techniques for generation of human monoclonal antibodies havealso been described but are generally more onerous than murinetechniques and not applicable to all antigens. See, e.g., Larrick etal., U.S. Pat. No. 5,001,065, for review (incorporated by reference forall purposes). One technique that has successfully been used to generatehuman monoclonal antibodies against a variety of antigens is the triomamethodology of Ostberg et al. (1983), Hybridoma 2:361-367, Ostberg, U.S.Pat. No. 4,634,664, and Engleman et al., U.S. Pat. No. 4,634,666(incorporated by reference for all purposes). The antibody-producingcell lines obtained by this method are called triomas, because they aredescended from three cells—two human and one mouse. Triomas have beenfound to produce antibody more stably than ordinary hybridomas made fromhuman cells.

An alternative approach is the generation of humanized immunoglobulinsby linking the CDR regions of non-human antibodies to human constantregions by recombinant DNA techniques. See Queen et al., Proc. Natl.Acad. Sci. USA 86:10029-10033 (1989) and WO 90/07861 (incorporated byreference for all purposes). The humanized immunoglobulins have variableregion framework residues substantially from a human immunoglobulin(termed an acceptor immunoglobulin) and complementarity determiningregions substantially from a mouse immunoglobulin, e.g., the L106antibody (see Example 1) (referred to as the donor immunoglobulin). Theconstant region(s), if present, are also substantially from a humanimmunoglobulin. The human variable domains are usually chosen from humanantibodies whose framework sequences exhibit a high degree of sequenceidentity with the murine variable region domains from which the CDRswere derived. The heavy and light chain variable region frameworkresidues can be derived from the same or different human antibodysequences. The human antibody sequences can be the sequences ofnaturally occurring human antibodies or can be consensus sequences ofseveral human antibodies. See Carter et al., WO 92/22653. Certain aminoacids from the human variable region framework residues are selected forsubstitution based on their possible influence on CDR conformationand/or binding to antigen. Investigation of such possible influences isby modeling, examination of the characteristics of the amino acids atparticular locations, or empirical observation of the effects ofsubstitution or mutagenesis of particular amino acids.

For example, when an amino acid differs between a murine L106 variableregion framework residue and a selected human variable region frameworkresidue, the human framework amino acid should usually be substituted bythe equivalent framework amino acid from the mouse antibody when it isreasonably expected that the amino acid:

(1) noncovalently binds antigen directly,

(2) is adjacent to a CDR region,

(3) otherwise interacts with a CDR region (e.g., is within about 3 Å ofa CDR region), or

(4) participates in the V_(L)-V_(H) interface.

Other candidates for substitution are acceptor human framework aminoacids that are unusual for a human immunoglobulin at that position.These amino acids can be substituted with amino acids from theequivalent position of the L106 antibody or from the equivalentpositions of more typical human immunoglobulins.

A further approach for isolating DNA sequences which encode a humanmonoclonal antibody or a binding fragment thereof is by screening a DNAlibrary from human B cells according to the general protocol outlined byHuse et Science 246:1275-1281 (1989) and then cloning and amplifying thesequences which encode the antibody (or binding fragment) of the desiredspecificity. The protocol described by Huse is rendered more efficientin combination with phage display technology. See, e.g., Dower et al.,WO 91/17271 and McCafferty et al., WO 92/01047. Phage display technologycan also be used to mutagenize CDR regions of antibodies previouslyshown to have affinity for ACT-4 receptors or their ligands. Antibodieshaving improved binding affinity are selected.

Anti-ACT-4 receptor antibodies that specifically bind to the sameepitope as the L106 antibody are usually identified by a competitivebinding assay. The assay has three components, an ACT-4 polypeptide(e.g., ACT-4-h-1), L106 antibody, which is usually labelled, and theantibody under test. Often the ACT-4 receptor polypeptide is immobilizedto a solid support. The test antibody binds to the same epitope as theL106 antibody if it reduces the amount of L106 antibody thatspecifically binds to the ACT-4 receptor polypeptide. The extent ofscreening necessary to obtain such antibodies can be reduced bygenerating antibodies with a protocol in which the specific epitopebound by L106 is used as an immunogen. Antibodies binding to the sameepitope as L106 may exhibit a substantially, but not completely,identical amino acid sequence to the L106 antibody, or may have anunrelated primary structure to the L106 antibody.

Anti-ACT-4 receptor antibodies having a different binding specificitythan L106 (i.e., which bind to a different epitope) are identified by acomplementary approach. Test antibodies are screened for failure tocompete with the L106 antibody for binding to an ACT-4 receptorpolypeptide. The extent of screening can be reduced by generatingantibodies with a protocol in which a fragment lacking a specificepitope bound by L106 is used as an immunogen.

Antibodies having the same or different binding specificity to aselected antibody to an ACT-4-L ligand polypeptide can be identified byanalogous procedures.

Antibodies having the capacity to stimulate or inhibit activation ofCD4⁺ or B cells can be identified by the screening procedures discussedin Section VI, infra. Some antibodies may selectively inhibit activationin response to some stimuli (e.g., alloantigenic but not mitogenic, orvice versa), and not to others. Some antibodies' inhibitory capacity maydepend on the time after activation at which the antibody is added. Someantibodies may have the capacity to activate CD4⁺ or B cellsindependently of other stimuli, whereas other antibodies may only havethe capacity to augment the efficacy of another stimulus such as thatprovided by PHA or PMA/ionomycin.

Antibodies isolated by the above procedures can be used to generateanti-idiotypic antibodies by, for example, immunization of an animalwith the primary antibody. For anti-ACT-4 receptor antibodies,anti-idiotype antibodies whose binding to the primary antibody isinhibited by ACT-4 receptors or fragments thereof are selected. Becauseboth the anti-idiotypic antibody and the ACT-4 receptors or fragmentsthereof bind the primary immunoglobulin, the anti-idiotypicimmunoglobulin may represent the “internal image” of an epitope and thusmay substitute for the ACT-4-L ligand polypeptide. Anti-idiotypicantibodies to an ACT-4-L ligand polypeptide that can substitute for anACT-4 receptor can be produced by an analogous approach.

C. Epitope Mapping

The epitope bound by the L106 antibody or any other selected antibody toan ACT-4 receptor is determined by providing a family of fragmentscontaining different amino acid segments from an ACT-4 receptorpolypeptide, such as ACT-4-h-1. Each fragment typically comprises atleast 4, 6, 8, 10, 20, 50 or 100 contiguous amino acids. Collectively,the family of polypeptide covers much or all of the amino acid sequenceof a full-length ACT-4 receptor polypeptide. Members of the family aretested individually for binding to e.g., the L106 antibody. The smallestfragment that can specifically bind to the antibody under testdelineates the amino acid sequence of the epitope recognized by theantibody. An analogous approach is used to map epitopes bound byantibodies to the ACT-4 ligand polypeptides.

D. Fragments of Antibodies, and Immunotoxins

In another embodiment of the invention, fragments of antibodies againstACT-4 receptors or their ligands are provided. Typically, thesefragments exhibit specific binding to the ACT-4 receptor or ligand withan affinity of at least 10⁷ M, and more typically 10⁸ or 10⁹ M. Antibodyfragments include separate heavy chains, light chains Fab, Fab′ F(ab′)₂,Fabc, and Fv. Fragments are produced by recombinant DNA techniques, orby enzymic or chemical separation of intact immunoglobulins.

In another embodiment, immunotoxins are provided. An immunotoxin is achimeric compound consisting of a toxin linked to an antibody having adesired specificity. The antibody serves as a targeting agent for thetoxin. See generally Pastan et al., Cell 47:641-648 (1986). A toxinmoiety is couple to an intact antibody or a fragment thereof by chemicalor recombinant DNA techniques. Preferably, the toxin is linked to animmunoglobulin chain in the form of a contiguous protein. See, e.g.,Chovnick et al., Cancer Res. 51:465; Chaudhary et al., Nature 339:394(1989) (incorporated by reference for all purposes). Examples ofsuitable toxin components are listed in Section I, supra, and arereviewed in, e.g., The Specificity and Action of Animal, Bacterial andPlant Toxins (ed. P. Cuatrecasas, Chapman Hall, London, 1976)(incorporated by reference for all purposes).

E. Hybridomas and Other Cell Lines

All hybridomas, triomas and other cell lines producing the antibodiesand their fragments discussed, supra, are expressly included in theinvention. These include the hybridoma line HBL106, deposited under theBudapest Treaty at the American Type Culture Collection, Rockville, Md.20852 as ATCC HB11483, which produces the L106 mouse antibody.

F. Uses of Antibodies

Antibodies to ACT-4 and ACT-4-L polypeptides and their binding fragmentsare useful for screening cDNA expression libraries, preferablycontaining human or primate cDNA derived from various tissues and foridentifying clones containing cDNA inserts, which encodestructurally-related, immunocrossreactive proteins. See Aruffo & Seed,Proc. Natl. Acad. Sci. USA 84:8573-8577 (1987) (incorporated byreference for all purposes). Antibodies are also useful to identifyand/or purify immunocrossreactive proteins that are structurally orevolutionarily related to the native ACT-4 receptor or ACT-4-L ligandpolypeptides or to fragments thereof used to generate the antibody.Diagnostic and therapeutic uses of antibodies, binding fragmentsthereof, immunotoxins and idiotypic antibodies are described in SectionVII, infra.

VI. Screening for Agonists and Antagonists

ACT-4 and ACT-4-L polypeptides, fragments, analogs thereof, antibodiesand anti-idiotypic antibodies thereto, as well as other chemical orbiological agents are screened for their ability to block or enhancebinding of an ACT-4 to its ligand. In addition, they are tested fortheir ability to stimulate or inhibit metabolic processes, such as DNAsynthesis or protein phosphorylation in cells bearing either an ACT-4receptor or an ACT-4-L ligand polypeptide anchored to their surfaces.

In some methods, the compound under test is screened for its ability toblock or enhance binding of a purified binding fragment of an ACT-4receptor (or fusion protein thereof) to a purified binding fragment ofan ACT-4-L ligand polypeptide (or fusion protein thereof). In suchexperiments, either the receptor or ligand fragment is usuallyimmobilized to a solid support. The test compound then competes with anACT-4 or ACT-4-L fragment (whichever is not attached to the support) forbinding to the support. Usually, either the test compound or thecompeting ligand or receptor is labelled.

In other methods, either or both of the ACT-4 receptor and ACT-4-Lligand polypeptide, or binding fragments of these molecules, areexpressed on a cell surface. For example, an ACT-4-L ligand polypeptidemay be expressed on activated B-cells and/or an ACT-4 receptorpolypeptide expressed on activated CD4+ T-cells. Alternatively, eitherthe ligand or receptor can be expressed from recombinant DNA in e.g.,COS-7 cells (see Example 6). In these methods, the existence of agonismor antagonism is determined from the degree of binding between an ACT-4receptor and its ligand that occurs in the presence of the testcompound. Alternatively, activity of the test compound is assayed bymeasurement of ³H-thymidine incorporation into DNA or ³²P incorporationinto proteins in cells bearing an ACT-4 receptor and/or cells bearing anACT-4-L ligand polypeptide.

Compounds that block ACT-4 or ACT-4-L polypeptide-induced DNA synthesisor protein phosphorylation are antagonists. Compounds that activate DNAsynthesis or phosphorylation via interaction with an ACT-4 receptor orits ligands are agonists. Agonistic or antagonistic activity can also bedetermined from other functional or physical endpoints of leukocyteactivation, or from clinically desirable or undesirable outcomes, suchas cytolytic activity, or extravasation of leukocytes into tissues fromblood vessels.

The ability of agents to agonize or antagonize T-cell proliferation invitro can be correlated with the ability to affect the immune responsein vivo. In vivo activity is typically assayed using suitable animalmodels such as mice or rats. To assay the effect of agents on allograftrejection, for example, potential therapeutic agents can be administeredto the animals at various times before introduction of the allogeneictissue; and the animals can be monitored for graft rejection. Suitablemethods for performing the transplant and monitoring for graft rejectionhave been described (see, e.g., Hislop et al., J. Thorac. Cardiovasc.100:360-370 (1990)) (incorporated by reference for all purposes).

VII. Therapeutic and Diagnostic Methods and Compositions

A. Diagnostic Methods

Diseases and conditions of the immune system associated with an alteredabundance, or functional mutation, of an ACT-4 receptor or its mRNA, oran ACT-4-L ligand or its mRNA may be diagnosed using the probes and/orantibodies of the present invention. Detection of an ACT-4 receptor ormRNA allows activated CD4⁺ T-cells to be distinguished from otherleukocyte subtypes. For example, ACT-4 receptor can be detected using anantibody or an ACT-4-L polypeptide that specifically binds to the ACT-4receptor. The presence of activated CD4⁺ T-cells is indicative of a MHCclass II induced immune response against, e.g., invading bacteria.Comparison of the numbers of activated CD4⁺ cells and CD8⁺ cells mayallow differential diagnosis between bacterial and viral infections,which predominantly induce these respective activated cell types. Thepresence of activated CD4⁺ cells is also indicative of undesirablediseases and conditions of the immune system, such as allograftrejection, graft versus host disease, autoimmune diseases, allergies andinflammation. The efficacy of therapeutic agents in treating suchdiseases and conditions can be monitored.

Detection of ACT-4-L ligand or its mRNA may indicate the presence ofactivated B-cells and/or signal that the appearance of activated CD4⁺T-cells is imminent. ACT-4-L ligand can, for example, be detected usingan antibody or an ACT-4 receptor polypeptide that specifically binds tothe ACT-4-L ligand. Successive detection of ACT-4-L ligand followed byACT-4 receptor (or vice versa) can allow monitoring of a progressionthrough different temporal stages of activation in an immune response.

Diagnosis can be accomplished by removing a cellular sample (e.g., bloodsample, lymph node biopsy or tissue) from a patient. The sample is thensubjected to analysis for determining: (1) the amount of expressed ACT-4receptor or ACT-4-L ligand in individual cells of the sample (e.g., byimmunohistochemical staining of fixed cells with an antibody or FACS™analysis), (2) the amount of ACT-4 receptor or ACT-4-L ligand mRNA inindividual cells (by in situ hybridization with a labelled complementarypolynucleotide probe), (3) the amount of ACT-4 receptor or ACT-4-Lligand mRNA in the cellular sample by RNA extraction followed byhybridization to a labeled complementary polynucleotide probe (e.g., byNorthern blotting, dot blotting, solution hybridization or quantitativePCR), or (4) the amount of ACT-4 receptor or ACT-4-L ligand in thecellular sample (e.g., by cell disruption followed by immunoassay orWestern blotting of the resultant cell extract).

Diagnosis can also be achieved by in vivo administration of a diagnosticreagent (e.g., a labelled anti-ACT-4 or ACT-4-L antibody for monitoringof activated CD4⁺ T-cells or B-cells, respectively) and detection by invivo imaging. The concentration of diagnostic agent administered shouldbe sufficient that the binding to those cells having the target antigenis detectable compared to the background signal. Further, it isdesirable that the diagnostic reagent can be rapidly cleared from thecirculatory system in order to give the best target-to-background signalratio. The diagnostic reagent can be labelled with a radioisotope forcamera imaging, or a paramagnetic isotope for magnetic resonance orelectron spin resonance imaging.

A change (typically an increase) in the level of protein or mRNA of anACT-4 receptor or ACT-4-L ligand in a cellular sample from anindividual, which is outside the range of clinically established normallevels, may indicate the presence of an undesirable immune reaction inthe individual from whom the sample was obtained, and/or indicate apredisposition of the individual for developing (or progressing through)such a reaction. Protein or mRNA levels may be employed as adifferentiation marker to identify and type cells of certain lineages(e.g., activated CD4⁺ cells for the ACT-4 receptor) and developmentalorigins. Such cell-type specific detection may be used forhistopathological diagnosis of undesired immune responses.

B. Diagnostic Kits

In another aspect of the invention, diagnostic kits are provided for thediagnostic methods described supra. The kits comprise container(s)enclosing the diagnostic reagents, such as labelled antibodies againstACT-4 receptors and ACT-4-L ligands, and reagents and/or apparatus fordetecting the label. Other components routinely found in such kits mayalso be included together with instructions for performing thediagnostic test.

C. Pharmaceutical Compositions

The pharmaceutical compositions used for prophylactic or therapeutictreatment comprise an active therapeutic agent, for example, an ACT-4receptor, an ACT-4-L ligand, fragments thereof, and antibodies andidiotypic antibodies thereto, and a variety of other components. Thepreferred form depends on the intended mode of administration andtherapeutic application. The compositions may also include, depending onthe formulation desired, pharmaceutically-acceptable, non-toxic carriersor diluents, which are defined as vehicles commonly used to formulatepharmaceutical compositions for animal or human administration. Thediluent is selected so as not to affect the biological activity of thecombination. Examples of such diluents are distilled water,physiological saline, Ringer's solutions, dextrose solution, and Hank'ssolution. In addition, the pharmaceutical composition or formulation mayalso include other carriers, adjuvants, or nontoxic, nontherapeutic,nonimmunogenic stabilizers and the like.

D. Therapeutic Methods

The therapeutic methods employ the therapeutic agents discussed abovefor treatment of various diseases in humans or animals, particularlyvertebrate mammals. The therapeutic agents include ACT-4 receptors,binding fragments thereof, ACT-4-L ligands, binding fragments thereof,anti-ACT-4 receptor and anti-ACT-4-L ligand antibodies andanti-idiotypic antibodies thereto, binding fragments of theseantibodies, humanized versions of these antibodies, immunotoxins, andother agents discussed, supra. Some therapeutic agents function byblocking or otherwise antagonizing the action of an ACT-4 receptor withits ligand. Preferred therapeutic agents compete with the ligand forbinding to the receptor, or compete with the receptor for binding to theligand. Other therapeutic agents function by killing cells bearing apolypeptide against which the agent is targeted. For example, anti-ACT-4receptor antibodies with effector functions or which are conjugated totoxins, radioisotopes or drugs are capable of selectively killingactivated CD4⁺ T-cells. Similarly, anti-ACT-4-L ligand antibodies arecapable of killing activated B cells under analogous circumstances.Selective elimination of activated cells is particularly advantageousbecause an undesirable immune response can be reduced or eliminated,while preserving a residual immune capacity in the form of inactivatedB-cells, CD4⁺ cells and CD8⁺ cells to combat invading microorganisms towhich a patient may subsequently be exposed. Other therapeutic agentsfunction as agonists of the interaction between an ACT-4 receptor andACT-4-L ligand.

1. Dosages and Methods of Administration

In therapeutic applications, a pharmaceutical composition (e.g.,comprising an antibody to ACT-4-h-1 or ACT-4-L-h-1) is administered, invivo or ex vivo, to a patient already suffering from an undesirableimmune response (e.g., transplant rejection), in an amount sufficient tocure, partially arrest, or detectably slow the progression of thecondition, and its complications. An amount adequate to accomplish thisis defined as a “therapeutically effective dose” or “efficacious dose.”Amounts effective for this use will depend upon the severity of thecondition, the general state of the patient, and the route ofadministration, and combination with other immunosuppressive drugs, ifany, but generally range from about 10 ng to about 1 g of active agentper dose, with single dosage units of from 10 mg to 100 mg per patientbeing commonly used. Pharmaceutical compositions can be administeredsystemically by intravenous infusion, or locally by injection. Thelatter is particularly useful for localized undesired immune responsesuch as host versus graft rejection. For a brief review of methods fordrug delivery, see Langer, Science 249:1527-1533 (1990) (incorporated byreference for all purposes).

In prophylactic applications, pharmaceutical compositions areadministered to a patients at risk of, but not already suffering anundesired immune reaction (e.g., a patient about to undergo transplantsurgery). The amount of agents to be administered is a “prophylacticallyeffective dose,” the precise amounts of which will depend upon thepatient's state of health and general level of immunity, but generallyrange from 10 ng to 1 g per dose, especially 10 mg to 100 mg perpatient.

Because the therapeutic agents of the invention are likely to be moreselective and generally less toxic than conventional immunomodulatingagents, they will be less likely to cause the side effects frequentlyobserved with the conventional agents. Moreover, because some of thetherapeutic agents are human protein sequences (e.g., binding fragmentsof an ACT-4 receptor or ACT-4 ligand or humanized antibodies), they areless likely to cause immunological responses such as those observed withmurine anti-CD3 antibodies. The therapeutic agents of the presentinvention can be combined with each other. For example, a combination ofantibodies against an ACT-4-L ligand with antibodies against an ACT-4receptor is likely to provide a particularly effective blockade againstT-cell activation. The agents of the invention can also be combined withtraditional therapeutics, and can be used to lower the dose of suchagents to levels below those associated with side effects. For example,other immunosuppressive agents such as antibodies to the α3 domain, Tcell antigens (e.g., OKT4 and OKT3, CD28), B-cell antigen (B7 or B7-2),antithymocyte globulin, as well as chemotherapeutic agents such ascyclosporine, glucocorticoids, azathioprine, prednisone can be used inconjunction with the therapeutic agents of the present invention.

For destruction of a specific population of target cells, it can beadvantageous to conjugate the therapeutic agents of the presentinvention to another molecule. For example, the agents can be joined toliposomes containing particular immunosuppressive agents, to a specificmonoclonal antibody or to a cytotoxin or other modulator of cellularactivity, whereby binding of the conjugate to a target cell populationwill result in alteration of that population. A number of protein toxinshave been discussed supra. Chemotherapeutic agents include, for example,doxorubicin, daunorubicin, methotrexate, cytotoxin, and anti-sense RNA.Antibiotics can also be used. In addition, radioisotopes such asyttrium-90, phosphorus-32, lead-212, iodine-131, or palladium-109 can beused. The emitted radiation destroys the targeted cells.

2. Diseases and Conditions Amenable to Treatment

The pharmaceutical compositions discussed above are suitable fortreating several diseases and conditions of the immune system.

a. Transplant Rejection

Over recent years there has been a considerable improvement in theefficiency of surgical techniques for transplanting tissues and organssuch as skin, kidney, liver, heart, lung, pancreas and bone marrow.Perhaps the principal outstanding problem is the lack of satisfactoryagents for inducing immunotolerance in the recipient to the transplantedallograft or organ. When allogeneic cells or organs are transplantedinto a host (i.e., the donor and donee are different individual from thesame species), the host immune system is likely to mount an immuneresponse to foreign antigens in the transplant (host-versus-graftdisease) leading to destruction of the transplanted tissue. CD8⁺ cells,CD4⁺ cells and monocytes are all involved in the rejection of transplanttissues. The therapeutic agents of the present invention are useful toblock alloantigen-induced immune responses in the donee (e.g., blockageor elimination of allogen-activation of CD4⁺ T-cells by antibodies to anACT-4 receptor or ACT-4-L ligand) thereby preventing such cells fromparticipating in the destruction of the transplanted tissue or organ.

b. Graft Versus Host Disease

A related use for the therapeutic agents of the present invention is inmodulating the immune response involved in “graft versus host” disease(GVHD). GVHD is a potentially fatal disease that occurs whenimmunologically competent cells are transferred to an allogeneicrecipient. In this situation, the donor's immunocompetent cells mayattack tissues in the recipient. Tissues of the skin, gut epithelia andliver are frequent targets and may be destroyed during the course ofGVHD. The disease presents an especially severe problem when immunetissue is being transplanted, such as in bone marrow transplantation;but less severe GVHD has also been reported in other cases as well,including heart and liver transplants. The therapeutic agents of thepresent invention are used to block activation of, or eliminate, thedonor leukocytes (particularly activated CD4⁺ T-cells and B-cells),thereby inhibiting their ability to lyse target cells in the host.

c. Autoimmune Diseases

A further situation in which immune suppression is desirable is intreatment of autoimmune diseases such as insulin-dependent diabetesmellitus, multiple sclerosis, stiff man syndrome, rheumatoid arthritis,myasthenia gravis and lupus erythematosus. In these disease, the bodydevelops a cellular and/or humoral immune response against one of itsown antigens leading to destruction of that antigen, and potentiallycrippling and/or fatal consequences. Activated CD4⁺ T-cells are believedto play a major role in many autoimmune diseases such as diabetesmellitus. Activated B cells play a major role in other autoimmunediseases such as stiff man syndrome. Autoimmune diseases are treated byadministering one of the therapeutic agents of the invention, whichblock activation of, and/or eliminate CD4⁺ T-cells and/or B-cells.Optionally, the autoantigen, or a fragment thereof, against which theautoimmune disease is targeted can be administered shortly before,concurrently with, or shortly after the immunosuppressive agent. In thismanner, tolerance can be induced to the autoantigen under cover of thesuppressive treatment, thereby obviating the need for continuedimmunosuppression. See, e.g., Cobbold et al., WO 90/15152 (1990).

d. Inflammation

Inflammation represents the consequence of capillary dilation withaccumulation of fluid and migration of phagocytic leukocytes, such asgranulocytes and monocytes. Inflammation is important in defending ahost against variety of infections but can also have undesirableconsequences in inflammatory disorders, such as anaphylactic shock,arthritis, gout and ischemia-reperfusion. Activated T-cells have animportant modulatory role in inflammation, releasing interferon γ andcolony stimulating factors that in turn activate phagocytic leukocytes.The activated phagocytic leukocytes are induced to express a number ofspecific cells surface molecules termed homing receptors, which serve toattach the phagocytes to target endothelial cells. Inflammatoryresponses can be reduced or eliminated by treatment with the therapeuticagents of the present invention. For example, antibodies against ACT-4or ACT-4-L block activation of, or eliminate activated, CD4⁺ cells,thereby preventing these cells from releasing molecules required foractivation of phagocytic cell types.

e. Infectious Agents

The invention also provides methods of augmenting the efficacy ofvaccines in preventing or treating diseases and conditions resultingfrom infectious agents. Therapeutic agents having the capacity toactivate CD4⁺ T-cells and/or B-cells (e.g., certain monoclonalantibodies against an ACT-4 or ACT-4-L ligand) are administered shortlybefore, concurrently with, or shortly after the vaccine containing aselected antigen. The therapeutic agent serves to augment the immuneresponse against the selected antigen. These methods may be particularlyadvantageous in patients suffering from immune deficiency diseases.

f. HTLV-I Infections

Antibodies against ACT-4-h-L-1 are also useful for killingHTLV-1-infected cells. As noted above, such cells express a gp34 antigenthat is identical or nearly identical to ACT-4-h-1. These methods areusually performed in vivo or ex vivo on HTLV-1-infected individuals.However, the methods are also effective for killing HTLV-1-infected HIVcells in vitro. For example, the methods are particularly useful forprotecting hospital workers from infection through contact with tissuesamples under analysis. The risk of HTLV-I infection can be reduced bytreating the samples according to the present methods (provided ofcourse that the samples are being tested for something other than thepresence of HTLV-I).

Antibodies against ACT-4-h-L-1 and also antibodies against ACT-4-h-1 maybe effective to reduce or eliminate perturbations of the immune systemthat have been observed in individuals suffering from HTLV-I infection.Such perturbations may result from the presence of ACT-4-h-L-1 as asurface antigen on HTLV-I infected T-cells, which would allow theinfected T-cells to interact with CD4⁺ T-cells via an ACT-4 receptor.

g. Treatment of AIDS

HIV virus is known to infect human CD4⁺ T-cells by binding to the CD4receptor. However, it is likely that for productive infection to occurthe CD4 receptor must interact with another receptor present on thereceptor of CD4⁺ T-cells. The identification of ACT-4 as a receptor onthe surface of activated T-cells suggests that ACT-4 and/or its ligandACT-4-L may interact with CD4 and contribute to HIV infection ofT-cells. If so, therapeutically effective amounts of therapeutic agentstargeted against ACT-4 or ACT-4-L may be effective in aborting HIVinfection and thereby treating AIDS. These therapeutic agents may alsobe effective for killing HIV-infected CD4⁺ T-cells either in vivo or invitro. In vitro methods have utility in e.g. protecting hospital workersfrom accidental infection as discussed above.

The following examples are offered to illustrate, but not to limit, theinvention.

EXAMPLES Example 1 A Monoclonal Antibody Against ACT-4-h-1

Mice were immunized with PHA-transformed T-lymphoblasts. Splenocytesfrom immunized mice were fused with SP2/O myeloma cells and hybridomassecreting antibodies specific for the T-cell clone were selected. Thehybridomas were cloned by limiting dilution. A monoclonal antibody,designated L106, produced by one of the resulting hybridoma, wasselected for further characterization. The L106 antibody was found tohave an IgG1 isotype. A hybridoma producing the antibody, designatedHBL106 has been deposited as ATCC HB11483.

Example 2 Cellular Distribution of Polypeptide Recognized by L106Antibody

Samples containing the antibody L106 were made available to certainparticipants at the Fourth International Workshop and Conference onHuman Leucocyte Differentiation Antigens (Vienna 1989) for the purposeof identifying tissue and cell types which bind to the L106 antibody.The data from the workshop are presented in Leukocyte Typing IV (ed. W,Knapp, Oxford U. Press, 1989) (incorporated by reference for allpurposes) and an accompanying computer data base available from WalterR. Gilks, MRC Biostatistics Unit, Cambridge University, England. Thisreference reports the L106 antibody binds a polypeptide of about 50 kDa.This polypeptide was reported to be present on HUT-102 cells (atransformed T-cell line), PHA-activated peripheral blood lymphocytes, anEBV-transformed 8-lymphoid cell line, and HTLV-II transformed T-cellline, PMA-activated tonsil cells, ConA- or PHA-activated PBLs, andPMA-activated monocytes. The polypeptide was reported to besubstantially absent on inter alia resting basophils, endothelial cells,fibroblasts, interferon γ-activated monocytes, peripheral non-T-cells,peripheral granulocytes, peripheral monocytes, peripheral mononuclearcells, peripheral T cells, and peripheral red blood cells.

The present inventors have obtained data indicating that the 50 kDapolypeptide (hereinafter “ACT-4-h-1 receptor”) is preferentiallyexpressed on the CD4⁺ subspecies of activated T-cells. In one series ofexperiments, cell-specific ACT-4-h-1 expression was analyzed onunfractionated PBLs by a two-color staining method. PBL were activatedwith PHA for about two days (using the culture conditions described inExample 3), and analyzed for cell-surface expression of ACT-4-h-1 ondifferent cellular subtypes by staining with two differently-labelledantibodies (FITC and PE labels). Labels were detected by FACS™ analysisessentially as described by Picker et al., J. Immunol. 150:1105-1121(1993) (incorporated by reference for all purposes). One antibody, L106,was specific for ACT-4-h-1, the other antibody was specific for aparticular leukocyte subtype. FIG. 1 shows three charts in which L106staining is shown on the Y-axis of each chart, and anti-CD4, anti-CD8and anti-CD19 staining as the X-axes of the respective charts. For thechart stained with anti-CD4, many cells appear as double positives(i.e., express both CD4 and ACT-4-h-1). For the chart stained withanti-CD8, far fewer cells appear as double positives. For the chartstained with anti-CD19 (a B-cell marker), double-positive cells aresubstantially absent.

In another series of experiments expression of ACT-4-h-1 was analyzed bysingle-color staining on isolated cell types. Cells were stained withfluorescently, labelled L106 antibody and the label was detected byFACS™ analysis. See Engleman et al., J. Immunol. 127:2124-2129 (1981)(incorporated by reference for all purposes). In some experiments, cellswere activated by PHA stimulation for about two days (again using theculture conditions described in Example 3). The results from thisexperiment, together with those from the two-color staining experimentdescribed supra, are summarized in Table 1. Table 1 shows that about 80%of activated CD4⁺ cells expressed ACT-4-h-1 with a mean channelfluorescence of >20, irrespective whether the CD4⁺ cells are isolated(one-color staining) or in unfractionated PBLs (two-color staining). Thelevel of expression of ACT-4-h-1 on activated CD8⁺ cells is much lowerthan on activated CD4⁺ T-cells in the two-color staining experiment, andvery much lower in the one-color staining. Thus, the extent ofexpression on activated CD8⁺ cells appears to depend on whether the CD8+cells are fractionated from other PBLs before activation. Inunfractionated CD8⁺ cells (two-color staining), about 10% of cellsexpress ACT-4-h-1, with a mean channel fluorescence of about 4. In thefractionated cells, only about 4% of cells express ACT-4-h-1 with a meanchannel fluorescence of about 2. These data suggest that ACT-4-h-1 isexpressed only on a small subtype of activated CD8⁺ cells and that thissubtype is somewhat more prevalent when the CD8⁺ cells are activated inthe presence of other PBLs.

Table 1 also indicates that ACT-4-h-1 was substantially absent on allresting leukocyte subtypes tested (i.e., CD4⁺ T-cells, CD8⁺ T-cells,CD19⁺ B-cells, CD14⁺ monocytes, granulocytes and platelets), and wasalso substantially absent on activated B-cells and monocytes. ACT-4-h-1was also found to be substantially absent on most tumor cell linestested. However, Molt3, Raji and NC37 cell lines did show a low level ofexpression.

TABLE 1 CELL SPECIFICITY OF ACT-4-h-1 EXPRESSION Expression of ACT-4-h-1% Cells MCF¹ Two Color Staining CD4⁺ T-Cells (resting) <2 <2 CD4⁺T-Cells (activated)² 80 25 CD8⁺ T-Cells (resting) <2 <2 CD8⁺ T-Cells(activated) 10 4 CD19⁺ B-Cells (resting) <2 <2 CD19⁺ B-Cells (activated)<2 <2 CD14⁺ Monocytes (resting) <2 <2 CD14⁺ Monocytes (activated) <2 <2One Color Staining PBLs (resting) <2 3 PBLs (activated) 50 27 CD4⁺(resting) <2 <2 CD4⁺ (activated) 80 22 CD8⁺ (resting) <2 <2 CD8⁺(activated) 4 2 Granulocytes <2 <2 Platelets <2 <2 Tumor Lines Molt-4,CEM, Hut 78, H9, Jurkat <2 <2 HPB-ALL, Sezary, T-AU <2 <2 Molt-3 20 3B-LCL, Arent, RML, JY, KHY, PGf <2 <2 MSAB, CESS, 9037, 9062 <2 <2Dandi, Ramos, Namalwa <2 <2 Raji, NC37 30 4 U937, THP-1, HL-60 <2 <2Kgla, K562, HEL <2 <2 ¹MCF = Mean Channel Fluorescence. ²Cells indicatedas “activated” had been stimulated with PHA for about three days.

Example 3 Time Course of ACT-4-h-1 Expression Responsive to CD4⁺ T-cellActivation

CD4⁺ T-cells were tested for expression of ACT-4-h-1 receptors inresponse to various activating stimuli. CD4⁺ T-cells were purified fromperipheral blood mononuclear cells by solid-phase immunoadsorption(“panning”). 5×10⁴ CD4⁺ T-cells were cultured with an activating agentin microtiter wells containing RPMI medium supplemented with 10% humanserum. Three different activating agents were used: (1) 5×10⁴ irradiated(3000 rads) monocytes, (2) PHA (1 μg/ml) and (3) tetanus toxoid (5μg/ml). ³H-thymidine was added to the cultures 12-16 h before harvest.After harvest, cells were tested for the expression of cell surfaceantigens by incubation with various labelled antibodies (L106, anti-CD4and anti-CD8), as described by Engleman et al., J. Immunol.127:2124-2129 (1981).

FIG. 2 shows the appearance of ACT-4-h-1 in response to alloantigenactivation. Before activation, no expression was observed. Thepercentage of cells expressing the ACT-4-h-1 receptor increases withtime, peaking at about 30% after about seven days of alloantigenactivation. The results also show that essentially all cells expressingACT-4-h-1 also expressed the CD4 receptor and that essentially no suchcells expressed the CD8 receptor. FIG. 3 presents similar data for theappearance of ACT-4-h-1 in response to tetanus toxoid activation. Again,the percentage of cells expressing ACT-4-h-1 peaked at about seven days.However, at this time a higher percentage of cells (about 60%) expressedthe receptor. FIG. 4 presents similar data for the appearance ofACT-4-h-1 on CD4⁺ T-cells in response to PHA activation. In thissituation, the percentage of CD4⁺ T-cells expressing the receptor peaksat about 65% after three days of activation.

It is concluded that ACT-4-h-1 is a CD4⁺ T-cell activation antigen thatis expressed in response to diverse activating stimuli.

Example 4 Cloning ACT-4-h-1 cDNA

The cDNA clone for the ACT-4-h-1 receptor was isolated using a slightlymodified COS cell expression system, first developed by Aruffo & Seed,supra. RNA was isolated from 72-hour PHA activated human peripheralblood lymphocytes. Total RNA was extracted with TRI-reagent (MolecularResearch Center), and poly(A)+ RNA was isolated by oligo dT-magneticbead purification (Promega). cDNA was synthesized by the method ofGubler & Hoffman, Gene 25:263-369 (1982) using superscript reversetranscriptase (Gibco/BRL) and an oligo dT primer. The blunted cDNA wasligated to non-self-complementary BstX1 adaptors and passed over asephacryl S-400 spin column to remove unligated adaptors and smallfragments (<300 base pairs). The linkered cDNA was then ligated into aBstX1 cut eukaryotic expression vector, pcDNA-IRL, an ampicillinresistant version of pcDNA-I (Invitrogen). The precipitated and washedproducts of the ligation reaction were electroporated into E. colistrain WM1100 (BioRad). Plating and counting of an aliquot of thetransformed bacteria revealed a total count of 2 million independentclones in the unamplified library. Average insert size was determined tobe 1.2 kb. The bulk of the library was amplified in liquid culture, 250ml standard LB media. Plasmid was recovered by alkaline lysis andpurified over an ion-exchange column (Qiagen).

Sub-confluent COS-7 cells were transfected with the purified plasmid DNAby electroporation. Cells were plated on 100 mm dishes and allowed togrow for 48 hours. Cells were recovered from the plates with PBS-EDTAsolution, incubated with monoclonal antibody L106, and were pannedaccording to standard procedures. A second round panning revealedenrichment as numerous COS cells adsorbed to the plates. Episomal DNAwas recovered from the immunoselected cells by the Hirt method, andelectroporated into bacteria for amplification.

Bacteria transformed with plasmid from the second round Hirt preparationwere diluted into small pools of about 100 colonies. The pools wereamplified and their DNA purified and tested for the ability to conferexpression of the L106 antigen on COS-7 cells by immunofluorescence.Phycoerythrin-conjugated L106 antibody was used to stain COS-7 cellmonolayers and the cells were then examined by manual immunofluorescencemicroscopy. Miniprep DNA from four out of eight pools was positive whentested for expression. The pool with the best expression, pool E, wasdivided into smaller pools of 12 colonies. Three out of eight sub-poolswere positive, and sub-pool E1 was plated to allow for the analysis ofsingle colonies. Clone E1-27 was found to confer high level expressionof ACT-4-h-1 receptor on the surface of transfected COS cells.

Example 5 cDNA Sequence Analysis

The insert from the clone designated E1-27 was subcloned intopBluescript and sequenced by the dideoxy chain termination method, usingthe T7 polymerase autoread sequencing kit (Pharmacia) on an ALFsequencer (Pharmacia). Restriction mapping revealed several convenientsites for subcloning. Five subclones were generated in pBluescript andwere sequenced on both strands with M13 forward and universal primers.

The cDNA and deduced amino acid sequences of ACT-4-h-1 are shown in FIG.5. The ACT-4-h-1 cDNA sequence of 1,137 base pairs contains a 14-bp 5°untranslated region and a 209-bp 3′ untranslated region. An AATAAApolyadenylation signal is present at position 1,041 followed by an 80-bppoly A tail starting at position 1,057. The longest open reading framebegins with the first ATG at position 15 and ends with a TGA at position846. The predicted amino acid sequence is that of a typical type 1integral membrane protein. Hydrophobicity analysis revealed a putativesignal sequence following the initiating ATG, with a short stretch ofbasic residues followed by a longer stretch of hydrophobic residues. Apredicted signal peptide cleavage site is present at residue 22 or 24(the latter being the more likely by the criteria of von Heijne, NucleicAcids Res. 14:4683-4690 (1986)) (incorporated by reference for allpurposes), leaving a mature protein of 253 amino acid residues (or 255amino acids, if cleavage occurs at the less probable site).Hydrophobicity analysis also reveals a single large stretch of 27hydrophobic residues predicted to be the transmembrane domain, whichpredicts an extracellular domain of 189 (or 191) amino acids and anintracellular domain of 37 amino acids. The extracellular domain iscysteine rich, where 18 cysteines are found within a stretch of 135amino acids. The predicted molecular mass (Mr) for the mature protein is27,400, and there are two potential N-glycosylation sites at amino acidresidues 146 and 160.

Comparison of the amino acid sequence of ACT-4-h-1 with known sequencesin the swiss-port database using the BLAZE program reveals a sequencesimilarity with members of the nerve growth factor receptor superfamily.Amino acid sequences are at least 20% identical for NGF-R, TNF-R, CD40,41-BB, and fas/APO-1, and 62% for OX-40, allowing for gaps anddeletions. Alignments of the various proteins reveal the conservation ofmultiple cysteine rich motifs. Three of these motifs are present inACT-4-h-1 and OX-40, compared with four such motifs in NGF-R and CD40.

Comparison of the nucleotide sequence of ACT-4-h-1 with known sequencesin the Genbank and EMBL databases using the programs BLAST and FASTDBrevealed a high degree of sequence similarity with only one member ofthe nerve growth factor receptor family, OX-40. Allowing for gaps andinsertions, the sequence identity is 66%. Comparison of the ACT-4-h-1and OX-40 nucleotide sequences reveals that both contain a 14-bp 5′untranslated region, and both contain approximately 80-bp poly A tails.In ACT-4-h-1, however, there is a slight lengthening of the 3′untranslated region from 187-bp to 209-bp, and there is a lengthening ofthe coding region from 816-bp to 834-bp, a difference of 18-bp or 6amino acid insertions. Aligning the two amino acid sequences revealsthat four of the amino acid insertions occur prior to the signalsequence cleavage site. Thus, the mature ACT-4-h-1 receptor proteincontains one more amino acid residue than OX-40 (i.e., 253 vs. 252 aminoacids). Remarkably, the ACT-4-h-1 nucleotide sequence is much more GCrich, than the OX-40 sequence (70% v. 55%) indicating that the twosequences will not hybridize under stringent conditions.

Example 6 Production of Stable ACT-4-h-1 Transfectants

An XbaI-HindIII fragment was excised from the construct described inExample 4, and inserted into XbaI/HindIII-digested pcDNA-1-neo(Invitrogen) to generate an expression vector termed ACT-4-h-1-neo (FIG.6). This vector was linearized with Sf1 and electroporated into threeeukaryotic cell lines. These cell lines were SP2/O (a mouse myelomaderived from the Balb/c strain), Jurkat (a transformed human T-cellline) and COS-7 (an adherent monkey cell line). After a 48-h recoveryperiod, transformed cells were selected in 1 mg/ml G418 (Gibco). Afterthree weeks of selection, neo-resistant cell lines were incubated with asaturating concentration of L106 antibody, washed and overlayered onto100 mm petri dishes coated with goat anti-mouse IgG to select for cellsexpressing ACT-4-h-1. After washing off unbound cells, adherent cellswere recovered and expanded in tissue culture. Cell lines were subjectto two further rounds of panning and expression. The resulting celllines were shown by direct immunofluorescence staining to expressabundant ACT-4-h-1 (FIG. 7).

The same strategy and principles are used to obtain a stable cell lineexpressing ACT-4-L-h-1 (See Example 10).

Example 7 Production of an ACT-4-h-1-Immunoglobulin Fusion Protein

A soluble fusion protein has been constructed in which the extracellulardomain of ACT-4-h-1 is linked via its C-terminal to the N-terminal ofthe constant domain of a human immunoglobulin. The vector encodingACT-4-h-1 described in Example 4 was cleaved with SmaI and NotI toexcise all ACT-4-h-1 sequences downstream of the SmaI site including thetransmembrane, cytoplasmic and 3′ untranslated regions. The remainingregion encodes the soluble extracellular portion of ACT-4-h-1 (FIG. 8).The source of the immunoglobulin constant region to be joined to theACT-4-h-1 extracellular domain was a plasmid termed 5K-41BB-Eg1 (Proc.Natl. Acad. Sci. (USA) 89: 10360-10364) (incorporated by reference forall purposes). This plasmid contains a 1.3 kb BamHI/EagI genomicfragment encoding the hinge, CH2 and terminal CH3 domains of human Ig,isotype gamma 1. The fragment required modification for insertion intothe SmaI/NotI ends of the ACT-4-h-1 vector, while preserving the peptidereading frame across the SmaI junction to be formed by blunt-endligation. The vector 5k-41BB-Eg1 was cut with BamHI and the resulting 5°extensions were filled with Klenow fragment. The vector was then cutwith EagI releasing the 1.3 kb fragment with blunt and NotI compatibleends. This fragment was ligated with SmaI/NotI digested ACT-4-h-1vector. The ligation mix was electroporated into E. coli and multipletransformant clones screened with PCR using ACT-4-h-1 and IgG1nucleotide fragments as primers.

Plasmids containing the ACT-4-h-1-IgG1 coding were electroporated intoCOS cells. The cells were allowed to grow for five days at which pointtheir supernatants were harvested and sterile filtered through a 0.2micron membrane. The supernatants were tested for expression ofACT-4-h-1-IgG1 by dot blotting. Supernatants were blotted ontomitrocellulose and blocked with 5% nonfat dry milk. Replica blots wereprobed with antibody L106 or alkaline phosphatase-labelled goatanti-human immunoglobulin IgG (American Qualex). Antibody L106 wasdetected with an alkaline phosphatase labelled goat anti-mouse IgG.NBT/BCIP (Pierce) was used as a colorimetric substrate. High producingpositive clones were sequenced at the junction site to confirm propervector construction. The resulting fusion gene is depicted in FIG. 9.

Example 8 Identification of Cells Types Expressing a Ligand to ACT-4-h-1

Cell types expressing a ligand to ACT-4-h-1 were identified by indirectstaining combined with flow cytometry using the ACT-4-h-1 recombinantimmunoglobulin fusion protein described in Example 7 (hereinafterACT-4-h-1-Rg) as a probe. Bound fusion protein was detected usingphycoerythrin-conjugated anti-human Ig. These experiments revealed thata ligand to ACT-4-h-1 was expressed at low levels on a few B-cell lines,including a Burkitt lymphoma cell line (Jiyoye), and the EBV-transformedLCL's 9037, 9059, and MSAB. These cell lines scored 5% positive with aMCF of 3. See Table 2. With the cell line Jiyoye, it was possible toenrich for cells staining positive by the panning procedure to yield acell line Jo-P5, which stained 90% positive with a MCF of 10. Other celllines tested, including PBMC's and purified subpopulations of freshlyisolated T-cells, B-cells, monocytes, dendritic cells, most T-cell tumorlines and myelomonocytic tumor cell lines, were substantially absent ofa ligand to ACT-4-h-1. However, two HTLV-I infected T cell lines,HUT-102 and MT-2 expressed ACT-4-L-h-1, the latter at extremely highlevels.

The experiment was repeated after activation of cells using either PHAor a combination of PMA/ionomycin. PBMC's activated with 2 μg/ml of PHAfor three days stained between 2-10% positive for a ligand to ACT-4-h-1,depending on the donor. Activation of B-LCL cells and Burkit lymphomalines using a combination of PMA (10 ng/ml) and ionomycin (500 ng/ml)induced substantial expression of ligand for some cell lines,particularly those such as MSAB that showed low levels of expression inthe resting state. See Table 2. Unfractionated B cells also showedpreferential expression of ligand (15% cells positive, MHC=5). Timecourse studies on MSAB cells indicated that ligand expression begins onday 2 following activation, and peaks on day 3 or 5.PMA/ionomycin-activation also induced preferential expression of ligandin erythroleukemia cell lines and in one of the three myelomonocyticcell lines tested, THP-1. PMA/ionomycin activation also induced a lowlevel of expression of ligand in the T-cell lines but not in the otherT-cell lines tested.

TABLE 2 Expression of Ligand to ACT-4-h-1 on Different Cell Types TUMORCELL LINE SCREEN Resting Activated¹ B-LCL MSAB 2-5%² mcf³ 4 80% mcf 21CESS <2 70% mcf 25 JY <2 40% 9 REM <2 40% 11 9059 2-5%  mcf 4 40% 6 SKF<2 30% 3 9037 2-5%  mcf 4 25% 5 9062 <2 10% 5 PGF <2  6% 10 ARENT <2  4%5 KHY <2  3% 5 BURKIT Jiyoye 20% mcf 4  7% 5 LYMPHOMA Daudi <2 10% 5Naralwa <2  5% 5 Raji <2 <2 OTHER B CELL (Pre B) NC-37 <2 <2 (B-All) SB<2 15% 5 T-CELL HSB-2 <2 6% 3 Jurkat <2 <2 Mol +4 <2 <2 Mol +3 <2 <2HPB-ALL <2 <2 HU +78 <2 <2 H9 <2 <2 VB <2 <2 MYELO THP-1 <2 25% mcf 5MONOCYTIC V937 <2 <2 HL60 <2 <2 ERYTHRO HEL <2 25% 5 LEUKEMIA K562  2%25% 5 HTLV-I HUT-102 30% mcf 4 INFECTED MT-2 100%  100 T-CELLS¹PMA/ionomycin ²% positive cells ³Mean channel fluorescence

Example 9 Cloning cDNA Encoding an ACT-4-h-1 Ligand

The cDNA clone for the ligand was isolated using a slightly modified COScell expression system, first developed by Aruffo & Seed, supra. RNA wasisolated from 72-hour PMA/ionomycin-activated human EBY-transformed Bcells (cell line MSAB). Total RNA was extracted with TRI-reagent(Molecular Research Center), and poly(A)+ RNA was isolated by oligodT-magnetic bead purification (Promega). cDNA was synthesized by themethod of Gubler & Hoffman, Gene 25:263-369 (1982) using superscriptreverse transcriptase (Gibco/BRL) and an oligo dT primer. The bluntedcDNA was ligated to non-self-complementary BstX1 adaptors and passedover a Sephacryl S-500 column to remove unligated adaptors and smallfragments (<300 base pairs). The linkered cDNA was then ligated into aBstX1 cut eukaryotic expression vector, pcDNA (Invitrogen). The ligationproducts were precipitated, washed and electroporated into E. colistrain MC1061/P3 generating an unamplified library of 100 millionindependent clones. The average insert size in the library was 1 kb. Thebulk of the library was amplified in 250 ml standard LB media. PlasmidDNA was recovered by alkaline lysis and purified over an ion-exchangecolumn (Qiagen).

Sub-confluent COS-7 cells were transfected with the purified plasmid DNAby electroporation. Cells were plated on 100 mm dishes and allowed togrow for 48 hours. Cells were recovered from the plates with PBS-EDTAsolution, incubated with monoclonal antibody ACT-4-h-1-Rg, and pannedaccording to standard procedures. A second round panning numerous COScells adsorbed to the plates showing enrichment for cells expressingligand. Episomal DNA was recovered from the immunoselected cells by theHirt method, and electroporated into bacteria for amplification.

Bacteria transformed with plasmid from the second round selection werecloned and amplified. DNA from individual clones was purified and testedfor the ability to confer expression of a ligand to ACT-4-h-1 in COS-7cells. Phycoerythrin-conjugated ACT-4-h-1-Rg was used to stain COS-7cell monolayers and the cells were then examined by manualimmunofluorescence microscopy. Clones #2, 26, and 30 gave high levelexpression of ACT-4-h-1-Rg binding activity.

Example 10 Sequence Analysis of a Ligand to ACT-4-h-1

The insert from clone 26 in Example 9 was subcloned into pBluescript atthe HindIII and XbaI cloning sites. The clone was sequenced by thedideoxy chain termination method, using a T7 polymerase-based autoreadsequencing kit (Pharmacia) on an ALF sequencer (Pharmacia). Threesubclones were generated in pBluescript and were sequenced on bothstrands with M13 forward and universal primers. The cDNA and predictedamino acid sequences of clone 26 are shown in FIG. 10. The polypeptideformed by the predicted amino acid sequence is designated ACT-4-L-h-1.

The ACT-4-L-h-1 cDNA sequence contains 1079 base pairs, with a 137 by 5′UTR and a 379 by 3′ UTR. An AATAAA polyadenylation signal is present atposition 1024 followed by a 20 base poly A tail beginning at position1049. Sequence analysis reveals a single open reading frame whichencodes a 183 amino acid polypeptide, with a calculated molecular weightof 21,000. The open reading frame begins with the first ATG at position149 and ends with a TGA at position 698. The ATG is flanked with a Kozakconsensus initiation sequence with an A position at −3 and a G at +4.Hydrophobicity analysis reveals that the predicted amino acid sequenceis that of a type II membrane protein with a single transmembrane domainof approximately 27 aa in the amino terminal portion of the protein.Also there are four N-linked glycosylation sites in the C-terminalportion of the molecule.

Comparison of the nucleotide sequence of ACT-4-L-h-1 with knownsequences in the Genbank and EMBL databases reveals no significanthomology to known genes except for a gene encoding a protein designatedgp34 by Miura et al., Mol. Cell. Biol. 11:1313-1325 (1991). The cDNAsequence of the coding region of the ACT-4-L-h-1 ligand is identical tothat of gp34. However, the ACT-4-L-h-1 cDNA contained an additional 112nucleotides at the 5′ end compared with the gp34 sequence. Because theadditional nucleotides occur within the 5′ untranslated region, theirpresence is unlikely to alter the expression product. Most likely, theACT-4-L-h-1 clone was derived from a more complete reverse transcriptand is more representative of the in vivo 5′ end. Possibly the extrasequence could be involved in regulating the translation of the protein.

Comparison of the predicted amino acid sequence of the ACT-4-L-h-1ligand with known sequences in the Protein Information Resource (PIR)database using the FastDB program revealed an identity with gp34, and avery weak homology with TNF alpha. A secondary structure predictionalgorithm developed by Chou & Fasman predicts that the ACT-4-L-h-1ligand and TNF-alpha are both likely to form significant amounts of betastructures. This prediction is consistent with the observation thatother members of the TNF family of proteins are all conformed orpredicted to form beta jelly roll configurations rich in betastructures.

For the purposes of clarity and understanding, the invention has beendescribed in these examples and the above disclosure in some detail. Itwill be apparent, however, that certain changes and modifications may bepracticed within the scope of the appended claims. All publications andpatent applications are hereby incorporated by reference in theirentirety for all purposes to the same extent as if each were soindividually denoted.

1. A purified ACT-4-L ligand polypeptide having a segment between 5 and160 contiguous amino acids from an amino acid sequence set forth in SEQID NO:4.
 2. The polypeptide of claim 1 that exhibits at least eightypercent sequence identity to the amino acid sequence of set forth in SEQID NO:4.
 3. The polypeptide of claim 2 having an antigenic determinantcommon to a protein with the amino acid sequence set forth in SEQ IDNO:4.
 4. The polypeptide of claim 3 comprising an intracellular domain,a transmembrane domain, or an extracellular domain.
 5. The polypeptideof claim 1 that comprises an extracellular domain of an ACT-4-L ligand.6. The polypeptide of claim 1 that is glycosylated.
 7. The polypeptideof claim 6 further comprising a linked second polypeptide.
 8. Thepolypeptide of claim 7, wherein the second polypeptide is animmunoglobulin constant domain.
 9. A purified extracellular domain of anACT-4-L ligand comprising at least five contiguous amino acids from thefull-length ACT-4-L-h-1 extracellular domain.
 10. The extracellulardomain of claim 9 that is full length.
 11. The extracellular domain ofclaim 9 that is in soluble form.
 12. The extracellular domain of claim11 that specifically binds to the ACT-4-L-h-1 ligand.
 13. Theextracellular domain of claim 11 that specifically binds to theACT-4-h-1 receptor.
 14. The extracellular domain of claim 13 consistingessentially of a domain that specifically binds to the ACT-4-h-1receptor.
 15. The extracellular domain of claim 9 that is labelled. 16.The extracellular domain of claim 9 that inhibits in vitro activation ofCD4⁺ T cells expressing the ACT-4-h-1 receptor on their surface.
 17. Theextracellular domain of claim 9 that stimulates in vitro activation ofCD˜+ T cells expressing the ACT-4-h-1 receptor on their surface.
 18. Theextracellular domain of claim 9 that competes with an antibodydesignated L106 for specific binding to the ACT-4-h-1 receptor.
 19. Theextracellular domain of claim 9 that competes with the ACT-4-L-h-1ligand for binding to an antibody.
 20. The extracellular domain of claim9 further comprising a linked second polypeptide.
 21. The extracellulardomain of claim 20, wherein the second polypeptide is a constant regionof an immunoglobulin heavy chain.
 22. An ACT-4 receptor polypeptideconsisting essentially of a domain that specifically binds to theACT-4-L-h-1 ligand. 23-50. (canceled)